Solid-phase purification of synthetic nucleic acid sequences

ABSTRACT

The invention provides a compound of the formula: 
     
       
         
         
             
             
         
       
     
     and a capture support of the formula: 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , R 3 , R 6 , A, B, D, E, J, K, Q, W, and Z are as defined herein. The invention also provides a method of purifying an oligonucleotide or an oligonucleotide analog composed of “b” nucleotides from a mixture comprising the oligonucleotide or oligonucleotide analog and at least one oligonucleotide or oligonucleotide analog composed of “a” nucleotides, wherein b≠a, comprising use of the compound and the capture support.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional patentapplication No. 62/356,214, filed on Jun. 29, 2016, which isincorporated herein by reference in its entirety.

FIELD

The application concerns a high-throughput method of purifying syntheticnucleic acid sequences utilizing a solid-phase support.

BACKGROUND

The ability to design and synthesize DNA and RNA sequences has had ahuge impact on biotechnology particularly in the rapidly growing fieldsof synthetic biology and nucleic acid-based drug development. Indeed,the use of synthetic DNA/RNA sequences and their analogues forrecognition and binding to messenger RNAs encoding disease-causingproteins has led to the production of nucleic acid-based drugs capableof inhibiting the expression of these proteins through either anantisense or an RNA interference pathway in the potential treatment awide spectrum of human diseases. Such applications require theproduction of synthetic nucleic acid sequences in large quantities(e.g., millimoles) and high purity for preclinical and clinicalinvestigations. In contrast, total gene synthesis for synthetic biologyapplications requires small amounts (e.g., nanomoles) of numerous highlypure synthetic DNA sequences. Although the chemical synthesis of nucleicacid sequences using the phosphoramidite chemistry is efficient and canbe scaled up for pharmaceutical production, the purification of thesesequences presents a formidable challenge.

Despite the fact that the coupling efficiency of phosphoramiditemonomers is near quantitative on controlled-pore glass (CPG) support,the full-length nucleic acid sequences are mixed with shorter sequencesresulting from incomplete phosphoramidite coupling at each cycle of thenucleic acid sequence assembly. Other process-related impurities consistof deletion sequences due to failure to quantitatively prevent thegrowth of shorter than full-length sequences and to completely removethe 5′-hydroxyl protecting group at each step of the nucleic acidsequence assembly. Furthermore, the formation of longer than full-lengthnucleic acid sequences occurs when the activation of phosphoramiditemonomers by a weak acid prompts the premature cleavage of theacid-labile 5′-hydroxyl protecting group of the newly extended nucleicacid sequence. Although these impurities are produced in small amounts,their physicochemical similarity to the desired nucleic acid sequencemakes them very difficult to remove.

In the context of large-scale nucleic acid-based drug production,HPLC-based methods including reversed-phase (RP) HPLC and anion exchangeHPLC are currently the preferred techniques for the purification ofnucleic acid sequences. The methods require high-capacity instrumentsand accessories (e.g., preparative columns) in addition to large volumesof buffered aqueous and organic elution solvents. This process isneither cost-effective nor amenable to parallel purification processes;only a single nucleic acid sequence can be purified per run unlessnumerous instruments are available for this purpose. HPLC-basedpurification processes are time-consuming given that, depending on thenature of individual nucleic acid sequence, more than one purificationrun may be required to achieve the level of sequence purity required forpharmaceutical applications. One important limitation of any large-scaleHPLC purification process is the burdensome removal of large volumes ofaqueous solvents produced during purification, which may also depend onthe physicochemical properties of each nucleic acid sequence; thisoperation requires costly equipment as well. In regard to thesmall-scale purification of DNA and RNA sequences for total geneconstruction in the realm of synthetic biology applications, HPLC-basedmethods can also be used for this purpose, but as discussed above, thesemethods are not amenable to cost-effective parallel purification ofnucleic acid sequences. Furthermore, HPLC-based purification methods aretime-consuming and often may not completely resolve shorter thanfull-length sequences from the desired nucleic acid sequences. Althoughpolyacrylamide gel electrophoresis (PAGE) can efficiently separateshorter nucleic acid sequences from full length sequences, recovery ofthe purified sequences from the gel matrix is cumbersome and laboriouswith limited potential for parallel purification of nucleic acidsequences. A number of orthogonal methods have, however, been proposedfor the purification of nucleic acid sequences. These methods are basedon affinity, hydrophobic or ion-pair chromatography and solid-phaseremoval of shorter than full-length nucleic acid sequences by enzymatichydrolysis or by hydrophobic retention of the full-length sequences.

All of those techniques are either not amenable to highly parallel orlarge scale purification of nucleic acid sequences or both. Thus, thereremains a need in the art for improved methods for the purification ofsynthetic DNA and RNA sequences.

SUMMARY

The invention provides a method of purifying an oligonucleotide or anoligonucleotide analog composed of “b” nucleotides from a mixturecomprising the oligonucleotide or oligonucleotide analog and at leastone oligonucleotide or oligonucleotide analog composed of “a”nucleotides, wherein b≠a, which method comprises:

(i) providing a protected nucleoside or nucleoside analog of formula (I)or (Ia) functionalized with an activatable phosphorus-containing entity:

wherein B is an optionally protected nucleobase or an optionallyprotected nucleobase analog,

D and E are independently C₂-C₁₀ alkanediyl,

n is 1 to 4,

R¹ is C₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl,

R² is hydrogen or linear or branched C₁-C₆ alkyl,

R³ is linear or branched C₃-C₆ alkyl,

J is H or OR⁷ wherein R⁷ is a reversible or permanent hydroxylprotecting group,

X is O or S,

Y is H or C₁-C₆ linear alkyl,

W is a lone pair of electrons or an oxo function,

when W is a lone pair of electrons, Z is NR⁴R⁵ wherein R⁴ and R⁵ areindividually C₁-C₆ alkyl, C₆₋₁₀ arylated C₁₋₆ alkyl, or C₃-C₈ cycloalkylor R⁴ and R⁵ taken together with the nitrogen atom to which they areattached form a saturated 3-10 membered heterocyclic ring optionallyincluding one or more additional heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur, and Q is OT wherein T is areversible or permanent hydroxyl protecting group, and

when W is an oxo function, Z is H and Q is O⁻,

(ii) providing a mixture comprising an optionally protected firstoligonucleotide or oligonucleotide analog V′ composed of b-1 nucleotidesor nucleotide analogs and having a free 5′-terminal OH group, whereinthe first oligonucleotide V′ comprises phosphate or phosphorothioatetriester linkages, or a combination thereof, and wherein the firstoligonucleotide V′ is linked at its 3′-terminus to a solid support,wherein at least one oligonucleotide or oligonucleotide analog composedof “a” nucleotides is also linked to the solid support,

(iii) coupling the first oligonucleotide V′ with the protectednucleoside or nucleoside analog of formula (I) or (Ia), to provide asecond oligonucleotide of the formula (II) or (IIa):

(iv) oxidizing or sulfurizing, optionally deprotecting, and cleaving thesecond oligonucleotide or oligonucleotide analog of the formula (II) or(IIa) from the solid support to form a mixture comprising a thirdoligonucleotide of the formula (III) or (IIIa):

wherein V is the moiety resulting after optional deprotection of thesecond oligonucleotide or oligonucleotide analog and wherein V is notlinked to the solid support,

(v) reacting the mixture comprising the third oligonucleotide oroligonucleotide analog of the formula (III) or (IIIa) with asilica-attached linker compound of the formula:

wherein A, K, and L are independently C₂-C₁₀ alkanediyl and R⁶ is H,C₁-C₆ linear or branched alkyl, or C₃-C₈ cycloalkyl, and wherein

is silica, to form a linker-attached oligonucleotide or oligonucleotideanalog of the formula (IV) or (IVa):

(vi) washing the linker-attached oligonucleotide of the formula (IV) or(IVa) with at least one solvent or mixture of solvents to remove theoligonucleotide(s) or oligonucleotide analog(s) composed of “a”nucleotides,

(vii) treating the linker-attached oligonucleotide or oligonucleotideanalog of formula (IV) or (IVa) with a desilylation agent, and

(viii) isolating the purified oligonucleotide or oligonucleotide analogcomposed of “b” nucleotides from the product of step (vii).

The invention also provides a compound of the formula:

wherein B is an optionally protected nucleobase or optionally protectednucleobase analog,

D and E are independently C₂-C₁₀ alkanediyl,

n is 1 to 4,

R² is hydrogen or C₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl,

R³ is linear or branched C₃-C₆ alkyl,

J is H or OR⁷ wherein R⁷ is a reversible or permanent hydroxylprotecting group,

X is O or S,

Y is H or linear C₁-C₆ alkyl,

W is a lone pair of electrons, and

Z is NR⁴R⁵ wherein R⁴ and R⁵ are individually C₁-C₆ alkyl, C₆-C₁₀arylated C₁-C₆ alkyl, or C₃-C₈ cycloalkyl containing 1 to 10 carbonatoms or R⁴ and R⁵ taken together with the nitrogen atom to which theyare attached form a saturated 3-10 membered heterocyclic ring optionallyincluding one or more additional heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur, and Q is OT wherein T is areversible or permanent hydroxyl protecting group.

The invention further provides a capture support of the formula (9):

wherein A, K, and L are independently C₂-C₁₀ alkanediyl and R⁶ is H,C₁-C₆ linear or branched alkyl, or C₁-C₆ cycloalkyl, and wherein

is silica.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the RP-HPLC profile of unpurified 10a (5′-functionalized12a).

FIG. 1B shows the RP-HPLC profile of the phosphorothioate DNA sequence10a after capture by the support 3.

FIG. 1C shows the RP-HPLC analysis of solid-phase purified 12a that hasbeen released from the support 11a.

FIG. 1D shows the purity analysis of the solid-phase purified 12a byPAGE.

FIG. 2A shows the RP-HPLC profile of unpurified 10b (5′-functionalized12b).

FIG. 2B shows the RP-HPLC profile of remaining unpurified 10b aftercapture by support 3.

FIG. 2C shows the RP-HPLC analysis of solid-phase purified 12b that hasbeen released from the support 11b.

FIG. 2D shows the purity analysis of the solid-phase purified 12b byPAGE.

FIG. 3A shows the RP-HPLC profile of unpurified 10c (5′-functionalized12c).

FIG. 3B shows the RP-HPLC profile of remaining unpurified 10c aftercapture by support 3.

FIG. 3C shows the RP-HPLC analysis of solid-phase purified 12c that hasbeen released from the support 11c.

FIG. 3D shows the purity analysis of the solid-phase purified 12c byPAGE.

FIG. 4A shows the RP-HPLC profile of unpurified 10d (5′-functionalized12d) spiked with a 14-, 16- and a 18-mer phosphorothioate DNA sequence,the ratio of 10d:14-mer:16-mer:18-mer being 5:1:1:1.

FIG. 4B shows the RP-HPLC profile of 10d that has been captured by thesupport 3.

FIG. 4C shows the RP-HPLC analysis of solid-phase purified 12d that hasbeen released from the capture support 11d.

FIG. 4D shows the purity analysis of the solid-phase purified 12d byPAGE. Left lane: Solid-phase capture of 10d spiked with RP-HPLC purified14-, 16- and 18-mer phosphorothioate DNA sequences. Middle lane:Solid-phase purified 12d. Right lane: Unpurified DNA sequence 10d spikedwith RP-HPLC purified 14-, 16- and 18-mer phosphorothioate DNAsequences.

FIG. 5A shows the RP-HPLC profile of unpurified 10e (5′-functionalized12e).

FIG. 5B shows the RP-HPLC profile of unpurified 10e after capture by thesupport 3.

FIG. 5C shows the RP-HPLC analysis of solid-phase purified 12e that hasbeen released from the support 11e.

FIG. 5D shows the purity analysis of the solid-phase purified 12e byPAGE.

FIG. 6A shows the RP-HPLC profile of unpurified 10f (5′-functionalized12f).

FIG. 6B shows the RP-HPLC profile of unpurified 10f after capture by thesupport 3.

FIG. 6C shows the RP-HPLC analysis of solid-phase purified 12f that hasbeen released from the support 11a.

FIG. 6D shows the purity analysis of the solid-phase purified 12f byPAGE.

FIGS. 7A and 7B show the RP-HPLC analysis of the enzymatic hydrolysis offully deprotected, solid-phase purified DNA sequence 12e and 12f,respectively, catalyzed by snake-venom phosphodiesterase (Crotallusadamanteus) and bacterial alkaline phosphatase (Escherichia coli).

FIG. 8 shows PAGE analysis of the purity of phosphorothioate DNAsequence 12a recovered from the solid-phase purification processdescribed herein and from the RP-HPLC purification process. Lane 1 showsunpurified 12a. Lane 2 shows PAGE analysis of solid-phase recovery of12a obtained from RP-HPLC-purified 10a. Lane 3 shows PAGE analysis ofrecovery of 12a from RP-HPLC-purified 10a that had been exposed to TBAF.

FIG. 9 shows the RP-HPLC chromatogram of silica gel-purified5′-silylated 2′-O-tert-butyldimethylsilyl uridine 13.

FIG. 10A shows the RP-HPLC profile of unpurified 10a (5′-functionalized12a) on 10-fold scale up.

FIG. 10B shows the RP-HPLC profile of remaining unpurified 10a aftercapture by support 3 on 10-fold scale up.

FIG. 10C shows the RP-HPLC analysis of solid-phase purified 12a that hasbeen released from the support 11a on 10-fold scale up.

FIG. 10D shows the purity analysis of the solid-phase purified 12a byPAGE on 10-fold scale up.

FIGS. 11A-11D shows RP-HPLC profiles that demonstrate the sequentialdeprotection of uracil-1-yl ribonucleoside 16a that is protected with a2′-O-iminooxymethyl propanoate protecting group.

FIG. 11A provides the RP-HPLC profile for the silica-gel-purified2′-O-protected uridine 16a.

FIG. 11B provides the RP-HPLC profile for the de-esterified2′-O-protected uridine 17a.

FIG. 11C provides the RP-HPLC profile for the 2′-O-deprotected uridine19a.

FIG. 11D provides the RP-HPLC profile for a commercial sample of uridineas a control.

FIGS. 12A-12D show RP-HPLC profiles that demonstrate the sequentialdeprotection of cytosine-1-yl ribonucleoside 16b that is protected witha 2′-O-iminooxymethyl propanoate protecting group.

FIG. 12A provides the RP-HPLC profile for the silica-gel-purified2′-O-protected cytosine 16b.

FIG. 12B provides the RP-HPLC profile for the de-esterified2′-O-protected cytosine 17b.

FIG. 12C provides the RP-HPLC profile for the 2′-O-deprotected cytosine19b.

FIG. 12D provides the RP-HPLC profile for a commercial sample ofcytosine as a control.

FIGS. 13A-13D show RP-HPLC profiles that demonstrate the sequentialdeprotection of adenine-9-yl ribonucleoside 16c that is protected with a2′-O-iminooxymethyl propanoate protecting group.

FIG. 13A provides the RP-HPLC profile for the silica-gel-purified2′-O-protected adenine 16c.

FIG. 13B provides the RP-HPLC profile for the de-esterified2′-O-protected adenine 17c.

FIG. 13C provides the RP-HPLC profile for the 2′-O-deprotected adenine19c.

FIG. 13D provides the RP-HPLC profile for a commercial sample of adenineas a control.

FIGS. 14A-14D show RP-HPLC profiles that demonstrate the sequentialdeprotection of guanine-9-yl ribonucleoside 16d that is protected with a2′-O-iminooxymethyl propanoate protecting group.

FIG. 14A provides the RP-HPLC profile for the silica-gel-purified2′-O-protected guanine 16d.

FIG. 14B provides the RP-HPLC profile for the de-esterified2′-O-protected guanine 17d.

FIG. 14C provides the RP-HPLC profile for the 2′-O-deprotected guanine19d.

FIG. 14D provides the RP-HPLC profile for a commercial sample of guanineas a control.

FIGS. 15A-15C provide RP-HPLC analysis of unpurified and desaltedchimeric RNA sequences. U: uracil-1-yl; T: thymin-1-yl.

FIG. 15A shows an RP-HPLC profile of an unpurified and desalted chimericRNA sequence of de-esterified 2′-O-protected (U_(p))₂₀dT.

FIG. 15B shows an RP-HPLC profile of an unpurified and desalted chimericRNA sequence of fully 2′-O-deprotected (U_(p))₂₀dT.

FIG. 15C shows an RP-HPLC profile of an unpurified and desalted chimericRNA sequence of a fully 2′-O-deprotected (U_(p))₂₀dT control sequence.

FIG. 16 shows the purity analysis of unpurified and desaltedde-esterified 2′-O-protected (U_(p))₂₀dT (lane 1), fully2′-O-deprotected (U_(p))₂₀dT (lane 2), and a fully 2′-O-deprotected(U_(p))₂₀dT control sequence (lane 3) RNA sequences, with bromophenolblue used as a marker and appearing as a large band at the bottom of thegel.

FIGS. 17A-17C provide RP-HPLC analysis of SVP/BAP hydrolysates. SVP:snake venom phosphodiesterase; BAP: bacterial alkaline phosphatase; Ura:uracil-1-yl; T: thymin-1-yl.

FIG. 17A shows an RP-HPLC analysis of an unpurified, de-esterified, anddesalted 2′-O-protected (U_(p))₂₀dT digest.

FIG. 17B shows RP-HPLC analysis of unpurified and desalted2′-O-deprotected (U_(p))₂₀dT digest.

FIG. 17C shows RP-HPLC analysis of unpurified 2′-O-deprotected(U_(p))₂₀dT control sequence digest.

FIG. 18 shows electrophoretic and RP-HPLC chromatographic data for RNAsequence 2′-OMe-r(UpCpApCpUpGpUpGpApApUpCpGpApUpGpCpCpApU), illustratingthat the disclosed method can successfully purify native RNA sequences.

FIG. 19 shows electrophoretic and RP-HPLC chromatographic data for RNAsequence2′-OMe-r(UpsCpsApsCpsUpsGpsUpsGpsApsApsUpsCpsGpsApsUpsGpsCpsCpsApsU),illustrating that the disclosed method can successfully purifyphosphorothioate RNA sequences.

SEQUENCE LISTING

The nucleic acid sequences listed in the accompanying sequence listingare shown using standard letter abbreviations for nucleotide bases, andthree letter code for amino acids, as defined in 37 C.F.R. 1.822. Onlyone strand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand. The Sequence Listing is submitted as an ASCII text file, createdon June 28, 2017, 7 KB, which is incorporated by reference herein.

DETAILED DESCRIPTION I. Definitions

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. When chemicalstructures are depicted or described, unless explicitly statedotherwise, all carbons are assumed to include hydrogen so that eachcarbon conforms to a valence of four. For example, in the structure onthe left-hand side of the schematic below there are nine hydrogen atomsimplied. The nine hydrogen atoms are depicted in the right-handstructure.

Sometimes a particular atom in a structure is described in textualformula as having a hydrogen or hydrogen atoms, for example —CH₂CH₂—. Itwill be understood by a person of ordinary skill in the art that theaforementioned descriptive techniques are common in the chemical arts toprovide brevity and simplicity to description of organic structures.

Referring now to terminology used generically herein, the term “alkyl”means a straight-chain or branched alkyl substituent containing from,for example, 1 to about 6 carbon atoms, preferably from 1 to about 4carbon atoms, more preferably from 1 to 2 carbon atoms. Examples of suchsubstituents include methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, and the like.

The term “cycloalkyl,” as used herein, means a cyclic alkyl substituentcontaining from, for example, about 3 to about 8 carbon atoms,preferably from about 4 to about 7 carbon atoms, and more preferablyfrom about 4 to about 6 carbon atoms. Examples of such substituentsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, and the like. The cyclic alkyl groups may be unsubstitutedor further substituted with alkyl groups such as methyl groups, ethylgroups, and the like.

The term “arylated alkyl” means an alkyl group substituted with anoptionally substituted aryl group, containing from, for example 6 to 10carbon atoms in the aryl group and 1 to about 6 carbon atoms in thealkyl group, preferably 6 carbon atoms in the aryl group and from 1 toabout 4 carbon atoms in the alkyl group, and more preferably 6 carbonatoms in the aryl group and from 1 to about 2 carbon atoms in the alkylgroup and wherein the alkyl group is connected to the rest of thecompound. Arylated alkyl has the same meaning as arylalkyl.

The term “heterocyclyl,” as used herein, refers to a monocyclic orbicyclic 5- or 6-membered ring system containing one or more heteroatomsselected from the group consisting of O, N, S, and combinations thereof.The heterocyclyl group can be any suitable heterocyclyl group andpreferably can be an aliphatic heterocyclyl group. The heterocyclylgroup can be a monocyclic heterocyclyl group or a bicyclic heterocyclylgroup. Suitable heterocyclyl groups include morpholine, piperidine,tetrahydrofuryl, oxetanyl, pyrrolidinyl, and the like. The heterocyclylor heteroaryl group is optionally substituted with 1, 2, 3, 4, or 5substituents as recited herein such as with alkyl groups such as methylgroups, ethyl groups, and the like, halo groups such as chloro, orhydroxyl groups, wherein the optional substituent can be present at anyopen position on the heterocyclyl group.

The term “aryl” refers to an unsubstituted or substituted aromaticcarbocyclic substituent, as commonly understood in the art, and the term“C₆-C₁₀ aryl” includes phenyl and naphthyl. It is understood that theterm aryl applies to cyclic substituents that are planar and comprise4n+2π electrons, according to Hückel's Rule.

The term “NR⁴R⁵” refers to a structure of the formula:

wherein, unless otherwise described, the R⁴ and R⁵ groups are eachindividually attached to the N atom and are not connected or otherwisebonded to each other.

The aminooxy function —ONH₂ includes both the free base and the acidaddition salts thereof. Non-limiting examples of suitable acids forformation of acid addition salts of aminooxy functions includehydrochloric acid, sulfuric acid, and the like.

The term “protecting group” refers to a group attached to a functionalgroup such as hydroxyl and/or that, when reversible, can be removed upontreatment with a suitable deprotecting agent. For example, a suitableprotecting group for a phosphoramidite can be a 2-cyanoethyl group.Examples of suitable protecting groups for hydroxyl groups are asfurther disclosed herein. A permanent hydroxyl protecting group is onewhich remains intact while subjected to subsequent reaction steps.Non-limiting examples of permanent hydroxyl protecting groups includebenzyl, methyl, and the like, which form benzyl and methyl ethers.

The terms “nucleoside” and “nucleotide” include moieties that containnot only the known purine and pyrimidine bases, e.g., adenine (A),thymine (T), cytosine (C), guanine (G), or uracil (U), but also otherheterocyclic bases or nucleobases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, or other heterocycles. Non-limiting examples ofsuch modifications include, for example, diaminopurine and derivativesthereof, inosine and derivatives thereof, alkylated purines orpyrimidines, acylated purines or pyrimidines, thiolated purines orpyrimidines, and the like, or modifications comprising addition of aprotecting group such as acetyl, difluoroacetyl, trifluoroacetyl,isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl,dimethylformamidine, dibutylformamidine, N,N-diphenyl carbamate,substituted thiourea, and the like. The purine or pyrimidine base mayalso be an analog of the foregoing. Those skilled in the art willrecognize suitable analogs that are described in the literature.Non-limiting example of typical analogs include, for example,1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine,8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queuosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine, and 2,6-diaminopurine.

The terms “nucleoside” and “nucleotide” further include moieties thatcontain not only conventional ribose and deoxyribose sugars andconventional stereoisomers, but other sugars as well, includingL-enantiomers and α-anomers. Modified nucleosides or nucleotides canalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms, aminegroups, or aliphatic groups, or are functionalized as esters, ethers,and the like. “Nucleotide analogs” and “nucleoside analogs” refer tomolecules having structural features that are recognized in theliterature as being mimetics, derivatives, having analogous structures,or other like terms, and include, for example, polynucleotides oroligonucleotides incorporating non-natural nucleotides, unnaturalnucleotide mimetics such as 2′-modified nucleosides including but notlimited to 2′-fluoro, 2′-O-alkyl, -O-alkylamino,-O-alkylalkoxy,protected-O-alkylamino,

—O-alkylaminoalkyl, —O-alkyl imidazole, and polyethers of the formula(O-alkyl)_(m) such as linear and cyclic polyethylene glycols (PEGs), and(PEG)-containing groups, locked nucleic acids (LNA), peptide nucleicacids (PNA), phosphorodiamidate morpholino oligomer (PMO) sequences,oligomeric nucleoside phosphonates, and any polynucleotide that hasadded substituent groups, such as protecting groups or linking groups.

As used herein, an activatable phosphorus-containing entity refers to aphosphoramidite or H-phosphonate group having suitable protective groupsand capable of being activated for formation of P—O bonds with the 3′-or 5′-hydroxyl group of a deoxyribonucleoside or deoxyribonucleosideanalog or ribonucleoside or ribonucleoside analog. In an embodiment, theactivatable phosphorus-containing entity can be a phosphoramidite groupor H-phosphonate group of the formula:

wherein W is a lone pair of electrons or an oxo function. When W is alone pair of electrons, Z is NR⁴R⁵ wherein R⁴ and R⁵ are individuallyC₁-C₆ alkyl, C₆-C₁₀ arylated C₁-C₆ alkyl, or C₃-C₈ cycloalkyl containing1 to 10 carbon atoms or R⁴ and R⁵ taken together with the nitrogen atomto which they are attached form a saturated 3-10 membered heterocyclicring optionally including one or more additional heteroatoms selectedfrom the group consisting of nitrogen, oxygen and sulfur, and Q is OTwherein T is a reversible or permanent hydroxyl protecting group. Theresulting phosphoramidite group has the structure:

When W is an oxo function, Z is H and Q is O⁻. The resultingH-phosphonate group has the structure:

The reversible hydroxyl protecting group can be any suitable reversiblehydroxyl protecting group. In a preferred embodiment, the reversiblehydroxyl protecting group is a 2-cyanoethyl group.

For RNA synthesis (J of formula I through IV or Ia through IVa is OR⁷),the 2′-hydroxy groups need to be protected with groups that can survivealkaline reaction conditions. The hydroxyl protecting groups can bereversible or permanent. The hydroxyl protecting groups can be anysuitable protecting groups including ethers, silyl ethers, benzylethers, substituted benzyl ethers, acetals, thioacetals, ketals,acetalesters, esters, orthoesters and the like. Examples of suitable2′-hydroxy protecting groups include t-butyldimethylsilyl (TBDMS),triisopropylsilyloxymethyl (TMM), or 2-cyanoethyloxymethyl (CEM) groups.

In some embodiments concerning RNA sequences, protection of the3′-hydroxyl may improve the efficiency of the 5′-functionalization.Certain bulky protecting groups, such as TBDMS, when used to protect the2′-hydroxyl, may block efficient protection at the 3′-hydroxyl.Accordingly, the 2′-hydroxyl protecting group may be selected to have areduced steric bulk, compared to bulky protecting groups such as TBDMS.Exemplary protecting groups include, but are not limited to acetals,ethers, benzyl ethers, substituted benzyl ethers, thioacetals, ketals,acetalesters, esters, or orthoesters. One exemplary protecting group hasa formula

With respect to this formula,

denotes the site of attachment to the hydroxyl, and R¹⁰ is H; alkyl,such as C₁₋₆alkyl, typically C₁₋₃alkyl, such as methyl, ethyl, n-propyl,or isopropyl; or cycloalkyl, such as C₃₋₈cycloalkyl; or OR¹⁰ is O⁻M⁺,where M⁺ is an alkali metal, such as K⁺, Na⁺, or Li⁺. In certainembodiments, R¹⁰ is ethyl.

II. Overview

The invention provides a method of purifying an oligonucleotide or anoligonucleotide analog composed of “b” nucleotides from a mixturecomprising the oligonucleotide or oligonucleotide analog and at leastone oligonucleotide or oligonucleotide analog composed of “a”nucleotides, wherein b≠a, which method comprises:

(i) providing a protected nucleoside or nucleoside analog of formula (I)or (Ia) functionalized with an activatable phosphorus-containing entity:

wherein B is an optionally protected nucleobase or an optionallyprotected nucleobase analog,

D and E are independently C₂-C₁₀ alkanediyl,

n is 1 to 4,

R¹ is C₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl,

R² is hydrogen or linear or branched C₁-C₆ alkyl,

R³ is linear or branched C₃-C₆ alkyl,

J is H or OR⁷ wherein R⁷ is a reversible or permanent hydroxylprotecting group,

X is O or S,

Y is H or C₁-C₆ linear alkyl,

W is a lone pair of electrons or an oxo function,

when W is a lone pair of electrons, Z is NR⁴R⁵ wherein R⁴ and R⁵ areindividually C₁-C₆ alkyl, C₆-C₁₀ arylated C₁-C₆ alkyl, or C₃-C₈cycloalkyl or R⁴ and R⁵ taken together with the nitrogen atom to whichthey are attached form a saturated 3-10 membered heterocyclic ringoptionally including one or more additional heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur, and Q is OT whereinT is a reversible or a permanent hydroxyl protecting group, and

when W is an oxo function, Z is H and Q is O⁻,

(ii) providing a mixture comprising an optionally protected firstoligonucleotide or oligonucleotide analog V′ composed of b-1 nucleotidesor nucleotide analogs and having a free 5′-terminal OH group, whereinthe first oligonucleotide V′ comprises phosphate or phosphorothioatetriester linkages, or a combination thereof, and wherein the firstoligonucleotide V′ is linked at its 3′-terminus to a solid support,wherein at least one oligonucleotide or oligonucleotide analog composedof “a” nucleotides is also linked to the solid support,

(iii) coupling the first oligonucleotide V′ with the protectednucleoside or nucleoside analog of formula (I) or (Ia), to provide asecond oligonucleotide of the formula (II) or (IIa):

(iv) oxidizing or sulfurizing, optionally deprotecting, and cleaving thesecond oligonucleotide or oligonucleotide analog of the formula (II) or(IIa) from the solid support to form a mixture comprising a thirdoligonucleotide of the formula (III) or (IIIa):

wherein V is the moiety resulting after optional deprotection of thesecond oligonucleotide or oligonucleotide analog and wherein V is notlinked to the solid support,

(v) reacting the mixture comprising the third oligonucleotide oroligonucleotide analog of the formula (III) or (IIIa) with asilica-attached linker compound of the formula:

wherein A, K, and L are independently C₂-C₁₀ alkanediyl and R⁶ is H,C₁-C₆ linear or branched alkyl, or C₃-C₈ cycloalkyl, and wherein

is silica, to form a linker-attached oligonucleotide or oligonucleotideanalog of the formula (IV) or (IVa):

(vi) washing the linker-attached oligonucleotide of the formula (IV) or(IVa) with at least one solvent or a mixture of solvents to remove theoligonucleotide(s) or oligonucleotide analog(s) composed of “a”nucleotides,

(vii) treating the linker-attached oligonucleotide or oligonucleotideanalog of formula (IV) or (IVa) with a desilylation agent, and

(viii) isolating the purified oligonucleotide or oligonucleotide analogcomposed of “b” nucleotides from the product of step (vii).

In certain preferred embodiments, A is 1,5-pentanediyl.

In certain preferred embodiments, K is 1,3-propanediyl.

In certain preferred embodiments, L is 1,3-propanediyl

In certain preferred embodiments, D is 1,2-ethanediyl.

In certain preferred embodiments, E is 1,5-pentanediyl.

In certain preferred embodiments, R² is methyl.

In certain preferred embodiments, R³ is 2-propyl.

In certain preferred embodiments, R⁶ is methyl.

In certain preferred embodiments, J is H.

In certain preferred embodiments, Y is methyl.

In certain preferred embodiments, the desilylation agent comprisesfluoride ion.

In certain particular embodiments, the protected nucleoside ornucleoside analog is of formula (I).

In certain embodiments, W is a lone pair of electrons. In certainembodiments, Z is NR⁴R⁵ wherein R⁴ and R⁵ are individually C₁ -C₆ alkyl,C₆-C₁₀ arylated C₁-C₆ alkyl, and C₃-C₈ cycloalkyl containing 1 to 10carbon atoms or R⁴ and R⁵ taken together with the nitrogen atom to whichthey are attached form a saturated 3-10 membered heterocyclic ringoptionally including one or more additional heteroatoms selected fromthe group consisting of nitrogen, oxygen, and sulfur. In certainembodiments, Q is OT wherein T is a reversible or permanent hydroxylprotecting group. In certain embodiments, T is 2-cyanoethyl. In certainembodiments, the compounds of formulas (I) and (Ia) have the structures:

In certain preferred embodiments, the optionally protected firstoligonucleotide or oligonucleotide analog is synthesized according to asolid-phase protocol.

The number of nucleotides “a” or “b” in the oligonucleotide oroligonucleotide analog can be from 2 to about 100,000, for example a orb can be from about 3 to about 50,000, from about 3 to about 25,000,from about 3 to about 10,000, from about 3 to about 5,000, from about 3to about 1,000, from about 3 to about 500, from about 5 to about 500,from about 10 to about 500, or from about 10 to about 250.

Desirably, the inventive method provides a purified oligonucleotide oroligonucleotide analog that is about 80% pure or more, e.g., about 81%pure or more, about 82% pure or more, about 83% pure or more, about 84%pure or more, about 85% pure or more, about 86% pure or more, about 87%pure or more, about 88% pure or more, about 89% pure or more, about 90%pure or more, about 91% pure or more, about 92% pure or more, about 93%pure or more, about 94% pure or more, about 95% pure or more, about 96%pure or more, about 97% pure or more, about 98% pure or more, or about99% pure or more. The purity of the oligonucleotide or oligonucleotideanalog can be expressed as a percentage based on the area under thecurve of an HPLC peak corresponding to the oligonucleotide oroligonucleotide relative to the total area under the curve for allcomponents. The purity of the oligonucleotide or oligonucleotide analogcan be determined using any suitable technique, for example, byreversed-phase HPLC or polyacrylamide gel electrophoresis (PAGE) withappropriate gel staining procedures and softwares for peak area or bandintensity measurements.

In accordance with an embodiment, the invention provides a compound ofthe formula (I):

wherein B is an optionally protected nucleobase or optionally protectednucleobase analog,

D and E are independently C₂-C₁₀ alkanediyl,

n is 1 to 4,

R¹ is C₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl,

R² is hydrogen or C₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl,

R³ is linear or branched C₃-C₆ alkyl,

J is H or OR⁷ wherein R⁷ is a reversible or permanent hydroxylprotecting group,

X is O or S,

Y is H or linear C₁-C₆ alkyl,

W is a lone pair of electrons, and

Z is NR⁴R⁵ wherein R⁴ and R⁵ are individually C₁-C₆ alkyl, C₆-C₁₀arylated C₁-C₆ alkyl, or C₃-C₈ cycloalkyl containing 1 to 10 carbonatoms or R⁴ and R⁵ taken together with the nitrogen atom to which theyare attached form a saturated 3-10 membered heterocyclic ring optionallyincluding one or more additional heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur, and Q is OT wherein T is areversible hydroxyl protecting group.

In certain preferred embodiments, D is 1,2-ethanediyl.

In certain preferred embodiments, E is 1,5-pentanediyl.

In certain preferred embodiments, R¹ is methyl.

In certain preferred embodiments, R² is methyl.

In certain preferred embodiments, R³ is 2-propyl.

In certain preferred embodiments, J is H. In other embodiments, J is OR⁷where R⁷ is a hydroxyl protecting group, and may have a formula

In certain preferred embodiments, Y is methyl.

In particular embodiments, the compound is:

In an embodiment, the invention provides a method for preparing thecompound of formula (I) comprising the steps of:

(i) providing a compound of the formula (1):

(ii) reacting the compound of step (i) with a compound of the formula:

R¹HN-D-NHR¹

to provide a compound of the formula (2):

(iii) reacting the compound of formula (2) with a compound of theformula:

to provide a compound of formula (3):

(iv) reacting the compound of formula (3) with a compound of theformula: (R³)₂SiX′₂, wherein X′ is a leaving group, to provide acompound of formula (4):

(v) reacting the compound of formula (4) with a nucleobase protected2′-deoxyribonucleoside or nucleobase-protected 2′-O-protectedribonucleoside or a nucleobase-protected and carbohydrate modifiedanalog thereof to provide a compound of the formula (5):

and

(vi) reacting the compound of formula (5) with [R⁴R⁵N]₂POCH₂CH₂CN orR⁴R⁵NP(X′)OCH₂CH₂CN wherein X′ is a monovalent leaving group to providea compound of the formula (6):

In accordance with an embodiment, the invention provides a capturesupport of formula (9):

wherein A, K, and L are independently C₂-C₁₀ alkanediyl and R⁶ is H,C₁-C₆ linear or branched alkyl, or C₁-C₆ cycloalkyl, and wherein

is silica.

In certain embodiments, R⁶ is methyl.

In certain embodiments, A is 1,5-pentanediyl.

In certain embodiments, K and L are individually 1,3-propanediyl.

In a particular embodiment, the compound is:

In an embodiment, the invention provides a method for preparing thecapture support of formula (9) comprising the steps of:

(i) providing a functionalized silica gel of the formula (10):

(ii) reacting the functionalized silica gel of the formula (10) with acompound of the formula:

to provide a compound of formula (11):

and

(iii) reacting the compound of formula (11) with a compound of theformula:

H₂NO-L-ONH₂

to provide the capture support of formula (9).

III. Chemistry

A simple and efficient process for the purification of phosphorothioateand native DNA and RNA sequences is described herein. An embodiment ofthis process consists of functionalizing commercial aminopropylatedsilica gel with aminooxyalkyl functions to enable capture of nucleicacid sequences carrying a 5′-siloxyl ether linker with a “keto” functionthrough an efficient oximation reaction. Deoxyribonucleosidephosphoramidites functionalized with the capture 5′-siloxyl ether linkerhave been prepared with yields in the range of 75-83% and incorporatedlast into the solid-phase assembly of DNA sequences. Capture of thenucleobase- and phosphate-deprotected DNA sequences released from thesynthesis support is demonstrated to proceed near quantitatively. Afterwashing off shorter than full length DNA sequences from the capturesupport, the purified DNA sequences are released from the support upontreatment with tetra-n-butylammonium fluoride in dry DMSO. The purity ofthe released DNA sequences is about or 98%. The scalability and highthroughput of the solid-phase purification process is demonstratedwithout sacrificing purity of the DNA sequences.

Phosphorothioate and native RNA sequences are prepared using a similarmethod. Typically, the 2′-hydroxyl is protected by a suitable protectinggroup, as described herein. FIGS. 18 and 19 provide electrophoretic andRP-HPLC chromatographic data for RNA sequences2′-OMe-r(UpCpApCpUpGpUpGpApApUpCpGpApUpGpCpCpApU) and2′-OMe-r(UpsCpsApsCpsUpsGpsUpsGpsApsApsUpsCpsGpsApsUpsGpsCpsCpsApsU),respectively, that were prepared and purified by the disclosed method.

A. Synthesis of Capture Support

3-aminopropyl-functionalized silica gel was identified as a suitablesupport for the intended purpose; the support is commercially availableand loaded with about 1 mmol of primary aminopropyl functions per gramof support. As shown in Scheme 1, the functionalization of theaminopropylated support began with the reaction of 7-oxooctanoic acid(1) with a coupling agent such as 1,1′-carbonyldiimidazole in anappropriate solvent such as THF to provide the ketoalkyl amidoalkylatedsupport 2; unreacted amino functions have been inactivated upon reactionwith excess acetic anhydride in dry pyridine.

The support 2 is then suspended in a solution of an appropriatebishydroxylamine such as O,O′-1,3-propanediylbishydroxylaminedihydrochloride in H₂O to provide the aminoxy-functionalized support 3.The selection of the aminooxy group for the functionalization of support3 is based on the high reactivity of this function by virtue of thewell-known a-effect with carbonyl groups. The concentration of aminooxyfunctions covalently bound to support 3 has been determined uponreaction of the 4-monomethoxytritylated ketone 6c with 3 and subsequentrelease of the 4-monomethoxytrityl cation under acidic conditions;spectrophotometric measurement of the yellow-colored cation at 478 nmreveals an aminooxy concentration of 146±7 μmoles per gram of support 3.

B. Synthesis of Linker Compound

As shown in Scheme 2, the linker compound 6c is prepared from the4-monomethoxytritylation of 6a, which was obtained from the reactionsequence 4a→5a. Butyrolactones 4a or 4b can be reacted with a suitablebis(methylamino)alkane such as N,N′-dimethylethylenediamine in water toform monoamides 5a or 5b. Acylation of amides 5a or 5b with a keto acidsuch as 7-oxooctanoic acid can be accomplished by reaction with adehydration reagent such as carbonyldiimidazole (CDI) in a suitablesolvent such as THF to provide keto diamides 6a or 6b. Tritylation of 6awith 4-monomethoxytrityl chloride in a suitable solvent such as drypyridine provides the tritylated compound 6c. The concentration ofaminooxy functions covalently bound to support 3 has been determinedupon reaction of the 4-monomethoxytritylated ketone 6c with 3 andsubsequent release of the 4-monomethoxytrityl cation under acidicconditions; spectrophotometric measurement of the yellow-colored cationat 478 nm reveals an aminooxy concentration of 146±7 μmoles per gram ofsupport 3. Although the reaction of aminooxy functions with carbonylsgroups have been shown to produce stable oxime ethers with a variety ofnucleosides and nucleic acid sequences, oximation reactions have notbeen used, to the best of our knowledge, for solid-phase purification ofnucleic acid sequences.

C. Synthesis of Protected Nucleoside or Nucleoside Analog of Formula (I)or (Ia) Functionalized with an Activatable Phosphorus-Containing Entity.

The conjugation of 6b to the 5′-hydroxy function of 2′-deoxythymidineand N-protected-2′-deoxyribonucleosides (7a-d) is performed through theformation of a diisopropylsiloxyl linkage upon reaction with equimolaramounts of dichlorodiisopropylsilane in the presence of imidazole andhas resulted in the production of the 5′-O-diisopropylsiloxyl etherderivatives 8a-d (Scheme 3) with yields in the range of 50% to 70% aftersilica gel purification. The alcohols 5a-b and 6a-b, the 4-methoxytritylether 6c and the silica gel-purified 5′-functionalizeddeoxyribonucleosides 8a-d have been fully characterized by ¹H-, ¹³C-NMRspectroscopies and by high resolution mass spectrometry (HRMS); thecharacterization data are presented in the Examples and figures. Thepurity of 8a-d has been assessed by RP-HPLC. It should be noted that tworotameric tertiary amides in each of 8a-d have contributed to the highcomplexity of their ¹H- and ¹³C-NMR spectra.

Phosphitylation of 8a-d (Scheme 3) is performed in a suitable solventsuch as anhydrous CH₂Cl₂ using commercial 2-cyanoethylN,N-diisopropylchlorophosphoramidite in the presence of a suitable basesuch as N,N-diisopropylethylamine. Given that silica gel purification ofthe nucleoside phosphosphoramites 9a-d is conducted in the presence oftriethylamine in order to prevent premature activation of thephosphoramidites when exposed to the inherent acidity of silica gel, itis advantageous that residual triethylamine be removed from 9a-d.Neutralization of 1H-tetrazole or any other acidic activator bytriethylamine will result in decreased coupling efficiency of thephosphoramidites during the course of solid-phase DNA synthesis.Lyophilization of frozen 9a-d benzene solutions, under high vacuum, hasbeen found effective for removing residual triethylamine fromphosphoramidite monomers. Triethylamine-free deoxyribonucleosidephosphoramidites (9a-d) have been isolated as viscous oils, the yieldsof which being in the range of 75-83%. These phosphoramidites have beensatisfactorily characterized by ³¹P NMR spectroscopy and HRMS; thecharacterization data are presented in the Experimental section. Asdiscussed above, the two rotameric tertiary amides within 8a-d have notonly contributed to the complexity of their ¹H- and ¹³C-NMR spectra buthave also added to the complexity of the diastereomeric ³¹P NMR signalsrecorded for 9a-d. It should also be emphasized that the presence ofadventitious moisture in 9a-d and commercial phosphoramidites willresult in lower phosphoramidite coupling efficiencies. It is thereforerecommended that all the deoxyribonucleoside phosphoramidites needed forsolid-phase synthesis of nucleic acid sequences be thoroughly dry, i.e.,dried overnight in a desiccator containing an efficient drying agent(e.g., phosphorus pentoxide) under high vacuum prior to use.

Consequently, comparing side-by-side the: (i) chemical shifts andmulticiplicity of the ¹H NMR signals; (ii) chemical shifts of the¹H-decoupled ¹³C- and ³¹P-NMR signals, where applicable, and (iii) HRMSdata appears to be the simplest approach to support reproducibility inthe preparation of 8a-d.

D. Solid-Phase Synthesis of 5′-Functionalized Phosphorothioate andNative DNA Sequences

The solid-phase synthesis of phosphorothioate and native DNA sequences(10a-d and 10e-f, respectively) can be conducted using commerciallong-chain alkylamine controlled-pore glass support (LCAA-CPG) accordingto standard protocols (see, for example, Iyer, R. P.; Phillips, L. R.;Egan, W.; Regan, J. B.; Beaucage, S. L. J. Org. Chem. 1990, 55,4693-4699) as shown in Scheme 4 with the following exception: thecapping step is performed after the oxidation reaction. The phosphitetriester function can then be oxidized using 0.05 M3H-1,2-benzodithiol-3-one 1,1-dioxide in MeCN or 0.02 M iodine solutionin THF/pyridine/water for phosphorothioate or native DNA sequences,respectively. It is advantageous that the coupling efficiency ofphosphoramidites 9a-d and the capping of unreacted 5′-hydroxy functionsbe optimal for solid-phase purification of DNA sequences; less thanoptimal coupling and capping reactions will result in poorer recovery ofsolid-phase-purified DNA sequences. In this regard, the coupling time ofDNA phosphoramidites 9a-d has been extended to 180 s to ensure thehighest coupling efficiency of these 5′-sterically-demandingphosphoramidite monomers. Post-synthesis deprotection and release of theDNA sequences 10a-f from LCAA-CPG have been performed under basicconditions according to standard protocols (see, for example, Ellington,A; Pollard, Jr., J. D. In Current Protocols in Molecular Biology; JohnWiley & Sons, New York, 1998, pp. 2.11.1-2.11.25).

E. Solid-Phase Capture and Release of Phosphorothioate and Native DNASequences.

Upon release from the LCAA-CPG support, the aqueous ammonia solutioncontaining the crude DNA sequence 10a-f and shorter than full lengthsequences (Scheme 4) is evaporated to half its original volume. Solidtetra-n-butylammonium chloride and the capture support 3 are thensequentially added to the aqueous solution of DNA sequences; thesuspension is kept at 65° C. over a period of 3 h. RP-HPLC analysis ofthe pre- and post-capture solutions of each DNA sequence shows that theoximation reaction resulting in the capture of each 5′-functionalizedDNA sequence is in all cases near complete. The solid support 11a-f isthen treated twice with a warm (55° C.) solution of aqueous ammonia inacetonitrile (Scheme 4, step 1) to wash off unbound shorter than fulllength DNA sequences by filtration. Exposure of the support 11a-f to 1.0M tetra-n-butylammonium fluoride (TBAF) in dry DMSO at 65° C. over aperiod of 3 h is sufficient to release the DNA sequences 12a-f fromtheir respective supports. The DNA sequences are isolated byprecipitation in dry THF and characterized by ESI-TOF MS. The purity ofthese sequences has been evaluated by RP-HPLC and by polyacrylamide gelelectrophoresis (PAGE) under denaturing conditions, as shown in FIGS. 1Cand 1D, 2C and 2D, 3C and 3D, 4C and 4D, 5C and 5D, and 6C and 6D, for12a-12f, respectively.

Efficiency of DNA sequence recovery from the solid-phase purificationprocess The most meaningful and reliable approach to measure efficiencyof the solid-phase purification of nucleic acid sequences is to use aspectrophotometrically measured amount of a RP-HPLC purified anddesalted 5′-functionalized DNA sequence (10a) and subject it to captureby the solid support 3 under the conditions described above. Uponexposure of the solid-support 11a to TBAF and subsequent precipitationof the released DNA sequence under the conditions reported in theexperimental section, the total amount of 12a, as measured by UVspectroscopy at 260 nm, is 90% the amount of 10a used for capture.

In order to assess whether the solid supports 3 and/or 11 did or did notdetrimentally affect the quality of the DNA sequence during the captureand release steps of the solid-phase purification process, the purity of12a has been evaluated by PAGE and compared to that of unpurified 12aand of 12a obtained directly from treatment of RP-HPLC-purified 10a withTBAF under conditions identical to those used for the release of 12afrom 11a. FIG. 8 clearly shows that purity of 12a that had beensubjected to the solid-phase purification process is highly comparableto the purity of 12a that had not been in contact with the capture solidsupport 3.

With the objective of demonstrating that the solid-phase purificationprocess can be qualified as highly parallel and scalable, thesolid-phase synthesis of ten identical phosphorothioate DNA sequences(10a) has been carried out, each on a scale of 1 μmole using theconditions described in Example 12. Upon completion of the syntheses,deprotection and release of each sequence from the CPG support, theammoniacal solution of each sequence is pooled together androtoevaporated to dryness under low pressure. The amounts of the capturesupport 3, reagents and solvent required for the capture of 10a havebeen increased by ten-fold while keeping the final concentration of thereagents the same as reported for individual syntheses. The capturereaction is performed under conditions identical to those describedabove (Scheme 3) for individual syntheses in terms of reaction time andtemperature. An aliquot of the capture reaction has been subjected toRP-HPLC analysis showing, as anticipated, near complete (>98%)disappearance of the DNA sequence 10a within 3 h (FIGS. 10A and 10B).Release of the DNA sequence 12a from the support 11a (see, e.g., FIG.10C) has been carried out using a 10-fold increase of 1.0 M TBAF/DMSOsolution that had been required for a 1 μmol scale reaction whilekeeping reaction time and temperature conditions the same. A 10-foldincrease in the volume of THF is necessary to precipitate 12a. The DNAprecipitate was isolated in a yield near proportional to the 1 μmolprocess scale, thereby conclusively demonstrating that the solid-phasepurification of nucleic acid sequences can be achieved in a highlyparallel manner and in yields comparable to those obtained from the 1μmol scale. ESI-MS analysis of the DNA precipitate has revealed a massconsistent with the theoretical molecular weight of the DNA sequence12a.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

IV. EXAMPLES

Materials and methods. All reactions sensitive to moisture and/or airare carried out under an atmosphere of argon in dry solvents underanhydrous conditions using oven-dried glassware. Common solvents(acetonitrile, benzene, dichloromethane, chloroform, methanol,2-propanol, hexane, acetone, ethyl acetate THF, formamide, DMF andDMSO), anhydrous solvents (acetonitrile, dichloromethane, THF, DMF,pyridine and DMSO), deuterated solvents (benzene-d₆ and DMSO-d₆) andchemicals including 7-oxooctanoic acid, 1,1′-carbonyldiimidazole,3-aminopropyl silica gel, acetic anhydride,1-methyl imidazole,O,O′-1,3-propanediylbishydroxylamine dihydrochloride, triethylamine,N,N-diisopropylethylamine, N,N′-dimethylethylenediamine, concentrated(28%) aqueous ammonia, 5,5-dimethyl-dihydro-furan-2-one, imidazole,dichlorodiisopropylsilane, methoxytrimethylsilane, 2-cyanoethylN,N-diisopropylchlorophosphoramidite, tetra-n-butylammonium chloride,tetra-n-butylammonium fluoride and anhydrous sodium sulfate were allpurchased from commercial sources and used without further purification.Ancillary reagents commonly used in solid-phase DNA synthesis including5′-O- and nucleobase-protected deoxyribonucleosides phosphoramidites,1H-tetrazole, 3H-1,2-benzodithiol-3-one 1,1-dioxide and succinylatedlong chain alkylamine controlled-pore glass (CPG) support functionalizedwith either 2′-deoxythymidine or N⁶-benzoyl-2′-deoxyadenosine, as theleader nucleoside, were obtained from reputable commercial sources andwere dried over fresh P₂O₅ in a dessicator under high vacuum prior touse. Reagents for enzymatic hydrolysis of native DNA sequences such asmagnesium chloride, Tris.Cl buffer, snake venom phosphodiesterase(Crotallus adamanteus) and bacterial alkaline phosphatase (E. coli) wereall purchased from commercial sources and used as received. Flashchromatography purifications are performed on glass columns (6.0 cm or2.5 cm I.D.) packed with silica gel 60 (EMD, 230-400 mesh), whereasanalytical thin-layer chromatography (TLC) analyses are conducted on 2.5cm×7.5 cm glass plates coated with a 0.25 mm thick layer of silica gel60 F₂₅₄ (EMD). Analytical RP-HPLC analyses are performed using a 5 μmSupelcosil LC-18S column (25 cm×4.6 mm) according to the followingconditions: starting from 0.1 M triethylammonium acetate pH 7.0, alinear gradient of 2.5% or 5.0% MeCN/min, when indicated, is pumped at aflow rate of 1 mL/min for 40 min. 2 M Triethylammonium acetate bufferwas purchased from Applied Biosystem and diluted to 0.1 M with HPLCgrade water prior to use. RP-HPLC-purified DNA sequences are desaltedusing commercial PD-10 (Sephadex G-25M) columns. All NMR experiments areperformed using a spectrometer operating at 300.13, 75.47 and 121.5 MHzfor one-dimensional ¹H, ¹H-decoupled ¹³C and ¹H-decoupled ³¹P,respectively. Samples are maintained at a temperature of 298° K. Allspectra are recorded in deuterated solvents or as indicated and chemicalshifts δ are reported in parts per million (ppm) relative to appropriateinternal references. High resolution mass spectra (HRMS) used to confirmthe elemental composition of new compounds were measured on a BrukerDaltonics ApexQ FTICR mass spectrometer equipped with a 12 T magnet.Electrospray ionization in positive ion mode was used to generate [M+H]⁺and [M+Na]⁺ ions out of test samples (0.01 mg dissolved in 1 mL of 10 mMammonium acetate in MeCN:H₂O (1:1 v/v)). Spectra were externallycalibrated using 0.5 mg/mL solution of CsI in water, which yielded aseries of peaks in the mass range used for analysis (200-2000 m/z).

Example 1

This example demonstrates a preparation of solid support 2.

To a solution of 7-oxooctanoic acid (1, 0.58 g, 3.0 mmol) in dry THF (4mL) was added 1,1′-carbonyldiimidazole (CDI, 0.49 g, 3.0 mmol); thesolution was stirred for 2 h at 25° C. Commercial 3-aminopropyl silicagel (1.0 g, about 1 mmol NH₂) was added to the solution after beingwashed with 20% triethylamine in MeCN (20 mL), filtered, and dried underargon. The suspension was mechanically shaken at 65° C. for 24 h. Afterfiltration, the solid support was washed successively with THF (20 mL)and MeCN (20 mL). The solid support was then suspended, over a period of30 min, in a commercial solution (20 mL) of acetic anhydride,1-methylimidazole and pyridine in THF to inactivate unreacted aminefunctions. After filtration, the support was washed with MeCN (2×20 mL)and then dried under high vacuum to give the functionalized solidsupport 2.

Example 2

This example demonstrates a preparation of solid support 3.

Solid support 2 (1.0 g) was placed in a 10 mL-glass vial to which wasadded a solution of O,O′-1,3-propanediylbishydroxylamine dihydrochloride(537 mg, 3.00 mmol) in H₂O (4 mL). The glass vial was sealed and thesuspension was mechanically shaken for 16 h at 65° C. The suspension wasfiltered, washed with DMF (20 mL), MeCN (20 mL) and dried under highvacuum to give the solid support 3, which was stored at −20° C. untilneeded. The concentration of aminooxy functions covalently attached to 3was measured by first washing the support (200 mg) with a solution (2mL) of Et₃N in MeCN (1:2 v/v) followed by MeCN (10 mL). The solidsupport (20 mg) was then added to a solution (300 μL) of 6c (60 mg. 0.1mmol) in DMSO:H20 (5:1 v/v). The suspension was mechanically agitated at25° C. over a period of 24 h and filtered. The 4-monomethoxytritylatedsupport was washed with MeCN (3×5 mL) and dried. Treatment of anaccurately measured amount of support with and accurate volume of 3%trichloroacetic acid in CH₂Cl₂, released the yellow-colored4-methoxytrityl cation, the absorbance of which wasspectrophotometrically measured at 478 nm to provide a surface densityof 146±7 μmoles of aminooxy functions per gram of support 3.

Example 3

This example demonstrates a synthesis of linkers which are used for thesolid-phase capture of DNA sequences or measuring the concentration ofaminooxy functions conjugated to support 3 in accordance with anembodiment of the invention.

4-Hydroxy-N-methyl-N-(2-(methylamino)ethyl)butanamide (5a).N,N′-dimethylethylenediamine (3.25 g, 40.0 mmol) and commercialy-butyrolactone (4a, 1.72 g, 20.0 mmol) were placed in a 25 mL-glassvial, which was sealed and heated at 90° C. for 16 h. The reactionmixture was then rotoevaporated to an oil under reduced pressure. Theoily material was loaded on the top of a glass column packed with silicagel (˜40 g) pre-equilibrated in CHCl₃:MeOH (9:1 v/v/v). The product waseluted from the column using a gradient of MeOH (10→20%) in CHCl₃ toafford 5a (2.32 g, 14.4 mmol) as an oil in a yield of 72%. ¹H NMR (300MHz, DMSO-d₆): δ 3.39 (dt, J=6.6, 2.0 Hz, 2H), 3.32 (dt, J=6.6, 2.0 Hz,2H), 2.95 (s, 1.5H), 2.79 (s, 1.5H), 2.59 (t, J=6.6 Hz, 1H), 2.53 (t,J=6.6 Hz, 1H), 2.34 (t, J=7.4 Hz, 1H), 2.29 (t, J=7.4 Hz, 1H), 2.28 (s,1.5H), 2.25 (s, 1.5H), 1.63 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ171.92, 171.91, 60.3, 49.7, 49.0, 48.9, 46.6, 36.2, 36.0, 35.4, 33.1,29.3, 28.6, 28.3, 28.0. +ESI-TOF MS: calcd for C₈H₁₈N₂O₂ [M+H]⁺175.1400,found 175.1405

4-Hydroxy-N,4-dimethyl-N-(2-(methylamino)ethyl)pentanamide (5b). Thepreparation of 5b has been performed at the same scale and conditionsused for the preparation of 5a with the exception of replacing 4a withcommercial 5,5-dimethyl-dihydro-furan-2-one (4b, 2.28 g, 20.0 mmol). Thepurification of 5b was carried out as described above for 5a andisolated as an oil (2.99 g, 14.8 mmol) in a yield of 74%. ¹H NMR (300MHz, DMSO-d₆): δ 4.23 (br s, 1H), 3.33 (q, J=6.6 Hz, 2H), 2.97 (s,1.5H),2.79 (s, 1.5H), 2.61 (t, J=6.6 Hz, 1H), 2.53 (t, J=6.6 Hz, 1.5H),2.37-2.27 (m, 3H), 2.28 (s, 15H), 2.25 (s, 1.5H), 1.57 (m, 2H), 1.08 (s,3H), 1.07 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 172.5, 172.4, 79.1,49.7, 49.0, 48.9, 46.6, 38.7, 38.3, 36.2, 35.9, 33.1, 28.9, 28.1, 27.4.+ESI-TOF MS: calcd for C₁₀H₂₂N₂O₂ [M+H]⁺203.1754, found 203.1763

N-(2-(4-hydroxy-N-methylbutanamido)ethyl)-N-methyl-7-oxooctanamide (6a).To a solution of 7-oxooctanoic acid (1, 2.45 g, 15.5 mmol) in dry THF(15 mL) was added 1,1′-carbonyldiimidazole (2.52 g, 15.5 mmol). Thesolution was stirred for 2 h at 25° C. and upon addition of 5a (2.32 g,14.4 mmol), the reaction mixture was allowed to stir at 65° C. for 24 h.The solution was rotoevaporated under reduced pressure; the materialleft was dissolved in CHCl₃ (40 mL) and vigorously mixed with water (20mL). The organic phase was collected and rotoevaporated to dryness underlow pressure. The crude product was then dissolved in a minimal volumeof CHCl₃ (4 mL) and loaded on the top of a glass column packed withsilica gel (˜40 g) pre-equilibrated in CHCl₃. The product 6a was elutedfrom the column using a gradient of MeOH (0→4%) in CHCl₃. Pure 6a wasisolated as an oil (3.57g, 11.3 mmol) in a yield of 78%. ¹H NMR (300MHz, DMSO-d₆): δ 4.44 (t, J=5 Hz, 1H), 3.46 (s, 0.7H), 3.39 (s, 3.5H),2.97 (s, 0.7H), 2.96 (s, 0.8H), 2.92 (s, 1.1H), 2.91 (s, 1.1H), 2.81 (m,2.1H), 2.40 (t, J=7.3 Hz, 2H), 2.32-2.15 (m, 4H), 2.07 (s, 3H), 1.62 (m,2H), 1.45 (m, 4H), 1.22 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 208.5,172.4, 172.3, 172.2, 172.1, 172.0, 171.9, 171.86, 171.81, 60.3, 60.24,60.20, 47.3, 46.5, 46.4, 45.8, 45.6, 44.2, 42.6, 35.9, 35.8, 35.1, 33.5,33.4, 33.12, 33.08, 32.4, 32.3, 31.4, 29.7, 29.3, 29.2, 28.4, 28.3,28.28, 28.20, 28.0, 27.9, 24.7, 24.6, 24.31, 24.26, 23.1. +ESI-TOF MS:calcd for C₁₆H₃₀N₂O₄ [M+Cs]⁺447.1255, found 447.1258.

N-(2-(4-hydroxy-N,4-dimethylpentanamido)ethyl)-N-methyl-7-oxooctanamide(6b) The preparation of 6b has been performed at the same scale andconditions used for the preparation of 6a with the exception ofreplacing 5a with 5b, (2.91 g, 14.4 mmol). The purification of 6b wascarried out as described above for 6a and isolated as an oil (3.97 g,11.6 mmol) in a yield of 80%. ¹H NMR (300 MHz, DMSO-d₆): δ 4.22 (m, 1H),3.47 (s, 0.4H), 3.39 (m, 3H), 3.33 (s, 0.4H), 2.98 (s, 0.6H), 2.96 (s,0.6H), 2.94 (s, 1H), 2.91 (s, 1H), 2.82 (d, J=2.1 Hz, 0.6H), 2.80 (s,1H), 2.40 (t, J=7.3 Hz, 2H), 2.33-2.21 (m, 3H), 2.17 (t, J=7.3 Hz,0.8H), 2.06 (s, 3H), 1.56-1.52 (m, 1.8H), 1.50-1.38 (m, 4.2H), 1.22 (m,2H), 1.08 (s, 1.4H), 1.06 (s, 3.8H). ¹³C NMR (75 MHz, DMSO-d₆): δ 208.4,172.9, 172.7, 172.5, 172.4, 172.2, 172.0,171.8, 171.7, 79.1, 68.25,68.21, 47.3, 47.2, 46.6, 46.4, 45.8, 45.6, 44.1, 44.0, 42.6, 38.7, 38.2,36.0, 35.8, 35.1, 35.0, 33.40, 33.37, 33.05, 33.01, 32.4, 32.3, 31.4,29.6, 29.2, 28.4, 28.3, 28.2, 28.14, 28.06, 27.2, 24.7, 24.6, 24.3,24.2, 23.1. +ESI-TOF MS: calcd for C₁₈H₃₄N₂O₄ [M+H]⁺343.2591, found343.2623.

N-(2-(4-((4-methoxyphenyl)diphenylmethoxy)-N-methylbutanamido)ethyl)-N-methyl-7-oxooctanamide(6c). To a solution of 6a (628 mg, 2.00 mmol) in dry pyridine was added4-methoxytrityl chloride (927 mg, 3.00 mmol). The reaction mixture wasstirred at 25° C. over a period of 4 h. The reaction was then quenchedupon addition of water (5 mL) and subjected to extraction using CHCl₃.Upon phase separation the organic phase was collected, dried overanhydrous sodium sulfate and filtered. The filtrate was concentratedunder reduced pressure to an oil. The oily material was loaded on thetop of a glass column packed with silica gel pre-equilibrated in asolution of CHCl₃:C₅H₅N (99.5:0.5 v/v). The reaction product was elutedfrom the column using a gradient of 0→2% MeOH in CHCl₃:C₅H₅N (99.5:0.5v/v). Fractions containing the product were collected and rotoevaporatedto dryness under low pressure. The material left was co-evaporated withtoluene (3×5 mL) to remove residual C₅H₅N. Pure 6c was isolated as anoil (1.01 g, 1.7 mmol) in a yield of 85%. ¹H NMR (300 MHz, DMSO-d₆): δ7.39-2.29 (m, 8H), 7.25-7.20 (m, 4H), 6.89 (d, J=8.8 Hz, 2H), 3.74 (s,3H), 3.42 (s, 0.5H), 3.36 (s, 2H), 2.98 (m, 2H), 2.93 (s, 0.7H), 2.90(s, 0.8H), 2.88 (s, 1H), 2.87 (s, 1H), 2.79 (m, 2H), 2.35 (m, 3H),2.26-2.12 (m, 3H), 2.04 (s, 1H), 2.03 (s, 1H), 2.02 (s, 1H), 1.77 (m,2H), 1.42 (m, 4H), 1.19 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 208.3,172.1, 172.0, 171.9, 171.7, 171.6, 171.5, 171.4, 158.1, 144.59, 144.57,135.4, 129.8, 128.8, 128.1, 127.9, 127.8, 126.7, 125.2, 113.1, 85.5,62.6, 62.5, 62.4, 55.0, 47.3, 47.2, 46.4, 46.3, 45.8, 45.6, 44.13,44.10, 42.6, 35.9, 35.8, 35.1, 35.0, 33.4, 33.3, 33.0, 32.34, 32.28,31.4, 29.58, 29.56, 29.3, 29.2, 28.38, 28.35, 28.3, 28.2, 25.4, 25.1,24.7, 24.5, 24.3, 24.2, 23.1, 21.0. +ESI-TOF MS: calcd for C₃₆H₄₆N₂O₅[M+Cs]⁺719.2456, found 719.2458.

Example 4

This example demonstrates a procedure for the preparation of5′-functionalized deoxyribonucleosides (8a-d) in accordance with anembodiment of the invention.

To 6b (780 mg, 2.28 mmol) and imidazole (184 mg, 2.70 mmol) in aflame-dried 25 mL-flask, was added under argon, dry DMF (5 mL) andN,N-diisopropylethylamine (2.35 mL,13.5 mmol); the solution was thencooled to 0° C. Dichlorodiisopropylsilane (730 μL, 4.50 mmol) was addedto the solution, which was left stirring for 1 h at 0° C. The reactionmixture was allowed to warm up to room temperature over 4 h and thencooled to −60° C. A solution of deoxyribonucleoside 7a (1.31 g, 5.40mmol) and imidazole (368 mg, 5.40 mmol) in dry DMF (5 mL) was addeddropwise to the reaction mixture, which was kept stirring at −60° C. for1 h. The reaction was then allowed to warm up to 0° C. and was leftstirring for 3 h at the same temperature. The reaction mixture wasquenched by the addition of cold (0° C.) 5% aq. NaHCO₃ (40 mL) and EtOAc(40 mL); after vigorous shaking, the organic layer was collected androtoevaporated to dryness under low pressure. The crude product wasdissolved in a minimal volume of CHCl₃ (4 mL) and loaded on the top of aglass column packed with silica gel (˜40 g) pre-equilibrated in CHCl₃.The product 8a was eluted from the column using a gradient of MeOH(0→6%) in CHCl₃. Pure 8a (1.32 g, 1.59 mmol) was isolated as a solid ina yield of 70%. The 5′-functionalized deoxyribonucleosides 8b-d havebeen similarly prepared, purified and isolated as oily materials inyields in the range of 50-68%.

8a: ¹H NMR (300 MHz, DMSO-d₆): δ 11.23 (s, 1H), 8.74 (s, 1H), 8.62 (s,0.7H), 8.59 (s, 0.3H), 8.32 (s, 1H), 8.07 (d, J=7.8 Hz, 2H), 7.65 (dd,J=7.6, 7.4 Hz, 1H), 7.56 (dd, J=7.6, 7.4 Hz, 2H), 6.51 (t, J=6.8 Hz,1H), 5.45 (m, 1H), 4.54 (bs, 1H), 3.98 (m, 2H), 3.85 (m, 1H), 3.38 (m,4H), 2.92 (m, 4H), 2.79 (m, 2H), 2.38 (t, J=7.3 Hz, 2H), 2.31-2.13 (m,4H), 2.05 (s, 3H), 1.66 (m, 2H), 1.43 (m, 4H), 1.21 (m, 7H), 0.97 (m,16H). ¹³C NMR (75 MHz, DMSO-d₆): δ 208.3, 172.4, 172.2, 172.1, 172.0,171.9, 171.8, 171.71, 171.66, 165.6, 151.9, 151.4, 150.3, 143.0, 142.8,133.4, 132.4, 128.5, 128.4, 125.9, 125.8, 87.2, 83.74, 83.66, 79.2,73.2, 73.1, 70.1, 70.4, 68.3, 68.2, 62.9, 62.7, 47.2, 46.5, 46.4, 45.8,45.7, 44.1, 44.0, 42.6, 38.5, 35.9, 35.8, 35.0, 33.5, 33.3, 33.2, 33.0,32.4, 32.3, 31.5, 29.6, 29.3, 29.2, 28.4, 28.3, 28.2, 28.1, 28.0, 27.3,27.1, 24.7, 24.6, 24.3, 24.2, 23.1, 17.60, 17.52, 17.47, 17.43, 17.3,17.21, 17.18, 17.11, 17.0, 12.9, 12.7, 12.6, 12.32, 12.29. +ESI-HRMS:calcd for C₄₁H₆₃N₇O₈Si [M+Na]⁺832.4399, found 832.4411.

8b: ¹H NMR (300 MHz, DMSO-d₆): δ 11.25 (s, 1H), 8.27 (d, J=7.3 Hz, 1H),8.01 (d, J=7.3 Hz, 2H), 7.63 (m, 1H), 7.52 (m, 2H), 7.37 (d, J=7.3 Hz,1H), 6.16 (t, J=6.2 Hz, 1H), 5.37 (d, J=4.4 Hz, 1H), 4.28 (m, 1H),4.01-3.89 (m, 3H), 3.47 (s, 0.4H), 3.38 (m, 3H), 2.98 (s, 0.7H), 2.95(s, 0.7H), 2.94 (s, 1.1H), 2.90 (s, 1.1H), 2.81 (m, 2H), 2.38 (m, 5H),2.23 (t, J=7.3 Hz, 2H), 2.15 (m, 2H), 2.05 (s, 3H), 1.70 (m, 2H), 1.43(m, 4H), 1.27 (s, 6H), 1.20 (m, 2H), 1.02 (m, 14H). ¹³C NMR (75 MHz,DMSO-d₆): δ 208.29, 208,27, 172.4, 172.2, 172.0, 171.9, 171.8, 171.70,171.66, 167.3, 163.0, 154.3, 144.3, 133.1, 132.7, 128.41, 128.38, 95.8,87.1, 86.0, 73.34, 73.30, 69.3, 62.3, 47.3 47.2, 46.5, 46.4, 45.8, 45.7,44.1, 44.0, 42.6, 40.8, 35.9, 35.8, 35.1, 35.0, 33.5, 33.3, 33.2, 33.0,32.4, 32.3, 31.5, 29.6, 29.39, 29.37, 28.4, 28.3, 28.2, 28.13, 28.07,27.3, 27.1, 24.7, 24.6, 24.31, 24.25, 23.1, 17.61, 17.59, 17.51, 17.48,12.8, 12.6. +ESI-HRMS: calcd for C₄₀H₆₃N₅O₉Si [M+Na]⁺808.4287, found808.4298.

8c: ¹H NMR (300 MHz, DMSO-d₆): δ 12.07 (s, 1H), 11.68 (s, 1H), 8.16 (s,1H), 6.22 (t, J=6.6 Hz, 1H), 5.37 (d, J=4.2 Hz, 1H), 4.42 (m, 1H), 3.88(d, J=7.8 Hz, 1H), 3.83 (m, 2H), 3.44-3.34 (m, 4H), 2.95 (s, 0.8H), 2.94(s, 0.8H), 2.91 (s, 1.2H), 2.89 (s, 1.2H), 2.77 (m, 3H), 2.63 (m, 2H),2.38 (t, J=7.3 Hz, 2H), 2.33-2.13 (m, 5H), 2.05 (s, 3H), 1.66 (m, 2H),1.43 (m, 4H), 1.21 (m, 7H), 1.12 (d, J=6.8 Hz, 6H), 0.95 (m, 14H). ¹³CNMR (75 MHz, DMSO-d₆): δ 208.3, 180.1, 172.4, 172.2, 172.1, 172.0,171.9, 171.8, 171.69, 171.66, 154.8, 148.3, 148.0, 137.2, 128.3, 120.3,87.1, 82.8, 73.2, 73.1, 70.0, 62.9, 47.3, 47.2, 46.5, 46.3, 45.7, 45.6,44.1, 44.0, 42.6, 35.9, 35.8, 35.05, 35.02, 34.7, 33.5, 33.3, 33.2,33.0, 32.4, 32.3, 31.4, 29.6, 29.3, 28.4, 28.3, 28.2, 28.1, 28.0, 27.2,27.1, 24.7, 24.5, 24.3, 24.2, 23.1, 18.83, 18.78, 17.55, 17.52, 17.48,17.43, 17.1, 12.7, 12.6. +ESI-HRMS: calcd for C₃₈H₆₅N₇O₉Si[M+Na]⁺814.4505, found 814.4518.

8d: ¹H NMR (300 MHz, DMSO-d₆): δ 11.33 (s, 1H), 7.42 (s, 1H), 6.17 (t,J=6.8 Hz, 1H), 5.31 (d, J=4.5 Hz, 1H), 4.25 (m, 1H), 3.85 (m, 3H), 3.46(s, 0.4H), 3.39 (s, 2 H), 3.36 (m, 1.1H), 2.97 (s, 0.8H), 2.95 (s,0.7H), 2.92 (s, 1H), 2.90 (s, 1H), 2.80 (m, 2H), 2.39 (t, J=7.3 Hz, 2H),2.13 (m, 1H), 2.22 (m, 2H), 2.12 (m, 3H), 2.06 (s, 3H), 1.76 (s, 3H),1.67 (m, 2H), 1.44 (m, 4H), 1.21 (m, 8H), 1.00 (m, 13H), 0.95 (s, 1H),0.93 (s, 1H). ¹³C NMR (75 MHz, DMSO-d₆): δ 208.3, 172.4, 172.2, 172.0,171.9, 171.8, 171.71, 171.67, 163.1, 150.3, 135.6, 109.4, 86.5, 83.6,73.3, 73.2, 70.0, 62.8, 47.3, 47.2, 46.5, 46.4, 45.8, 45.7, 44.1, 44.0,42.6, 35.9, 35.8, 35.1, 35.0, 33.5, 33.3, 33.1, 33.0, 32.4, 32.3, 31.5,29.6, 29.4, 28.4, 28.3, 28.2, 28.1, 28.0, 27.3, 27.1, 24.7, 24.6, 24.3,24.2, 23.1, 17.60, 17.56, 17.50, 17.48, 17.3, 12.8, 12.63, 12.59, 12.7.+ESI-HRMS: calcd for C₃₄H₆₀N₄O₉Si [M+Na]⁺719.4022, found 719.4031.

Example 5

This example demonstrates a general procedure for the preparation ofnucleoside phosphoramidites (9a-d) in accordance with an embodiment ofthe invention.

A solution of 8a (897 mg, 1.00 mmol) in CH₂Cl₂ (10 mL) was placed in aflame-dried 100 mL round-bottom flask. N,N-Diisopropylethylamine (536μl, 3.00 mmol) was added to the solution and followed by 2-cyanoethylN,N-diisopropylchlorophosphoramidite (335 μL,1.50 mmol). The reactionmixture was stirred at 25° C. for 3 h, quenched by the addition of H₂O(10 mL) and diluted with CH₂Cl₂ (25 mL). After vigorous shaking, theorganic layer was collected, dried over anhydrous Na₂SO₄ androtoevaporated under reduced pressure to afford an oil. The oilymaterial was evenly spread on the top of a column packed with silica gel(˜25 g) pre-equilibrated in benzene:Et₃N (9:1 v/v). The product waseluted from the column using benzene:Et₃N (9:1 v/v) as the eluent.Fractions containing the product were pooled together, concentratedunder reduced pressure and dissolved in dry benzene (7 mL). The solutionwas frozen and then lyophilized under high vacuum to affordtriethylamine-free 9a (750 mg, 0.83 mmol) as a colorless oil in 83%yield. The 5′-functionalized deoxyribonucleoside phosphoramidites 9b-dhave been similarly prepared and purified. Triethylamine-free 9b-d havebeen isolated as colorless oils, the yields of which are in the range of75-80%.

9a: ³¹P NMR (121 MHz, C₆D₆) δ 149.0, 148.8, 148.76, 148.72. +ESI-HRMS:calcd for C₅₀H₈₀N₉O₉PSi [M+H]⁺1010.5659, found 1010.5675.

9b: ³¹P NMR (121 MHz, C₆D₆) δ 148.3, 148.25, 148.22, 148.1, 147.91,147.89. +ESI-HRMS: calcd for C₄₉H₈₀N₇O₁₀PSi [M+H]⁺986.5546, found986.5548.

9c: ³¹P NMR (121 MHz, C₆D₆) δ 148.77, 148.73, 148.0, 147.92, 147.88.+ESI-HRMS: calcd for C₄₇H₈₂N₉O₁₀PSi [M+H]⁺992.5764, found 992.5773.

9d: ³¹P NMR (121 MHz, C₆D₆) δ 148.7, 148.6, 148.49, 148.46, 148.37.+ESI-HRMS: calcd for C₄₃H₇₇N₆O₁₀PSi [M+Cs]⁺1029.4257, found 1029.4266.

Example 6

This example demonstrates a solid-phase synthesis and deprotection ofthe DNA sequences 10a-f in accordance with an embodiment of theinvention.

Solid-phase synthesis of the phosphorothioate DNA sequences 10a[5′-d(A_(PS)C_(PS)A_(PS)C_(PS)T_(PS)G_(PS)T_(PS)G_(PS)A_(PS)A_(PS)T_(PS)C_(PS)G_(PS)A_(PS)T_(PS)G_(PS)C_(PS)C_(PS)A_(PS)T)](SEQ ID NO. 1), 10b[5′-d(C_(PS)T_(PS)C_(PS)C_(PS)G_(PS)T_(PS)A_(PS)C_(PS)C_(PS)T_(PS)T_(PS)A_(PS)C_(PS)G_(PS)T_(PS)C_(PS)T_(PS)T_(PS)G_(PS)T)](SEQ ID NO. 2), 10c[5′-d(G_(PS)T_(PS)G_(PS)A_(PS)G_(PS)T_(PS)A_(PS)G_(PS)C_(PS)G_(PS)A_(PS)A_(PS)C_(PS)G_(PS)T_(PS)G_(PS)A_(PS)A_(PS)G_(PS)T)](SEQ ID NO. 3), 10d[5′-d(T_(PS)A_(PS)T_(PS)C_(PS)C_(PS)G_(PS)T_(PS)A_(PS)G_(PS)C_(PS)T_(PS)A_(PS)A_(PS)C_(PS)G_(PS)T_(PS)C_(PS)A_(PS)G_(PS)T)](SEQ ID NO. 4), 10e[5′-d(A_(P)C_(P)A_(P)C_(P)T_(P)G_(P)T_(P)G_(P)A_(P)A_(P)T_(P)C_(P)G_(P)A_(P)T_(P)G_(P)C_(P)C_(P)A_(PT))](SEQ ID NO. 5) and 10f [5′-d(T_(d)T_(P)C_(p)A_(P)C_(P)T_(P)G_(p)T_(P)G_(P)A_(P)A_(P)T_(P)C_(P)G_(P)A_(P)T_(P)G_(P)C_(P)A_(P)A_(P)T_(P)G_(P)C_(P)C_(P)T_(p)G_(P)T_(P)G_(P)A_(P)A_(P)T_(P)C_(P)G_(P)A_(P)T_(P)C_(P)C_(p)A_(P)T_(P)C_(P)A_(P)C_(P)T_(P)G_(P)T_(P)G_(P)A_(P)A_(P)T_(P)C_(P)G_(P)A_(P)T_(P)G_(P)C_(P)C_(P)A_(P)T)](SEQID NO. 6) was conducted on a scale of 1 μmole using a succinyl longchain alkylamine controlled-pore glass (500 Å LCAA-CPG) or (2000ÅLCAA-CPG for 10f) support functionalized with5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine as the leader nucleoside.The syntheses have been carried out using a DNA/RNA synthesizer andcommercial 5′-O-(4,4′-dimethoxytrityl)-dA^(Bz), -dG^(iBu), -dC^(Bz), -dTphosphoramidite monomers, which have been each dissolved in dry MeCN togive a 0.1 M solution. The modified deoxyribonucleoside phosphoramidites9a-d have also been dissolved in dry MeCN to each provide a 0.15 Msolution. Each solution was placed in a vial connected to the DNA/RNAsynthesizer through an additional delivery port. Commercial 1H-tetrazolesolution was used for phosphoramidite activation in the solid-phasesynthesis of 10a-f. The reaction times of the coupling, capping andoxidation steps in the synthesis of the native and phosphorothioate DNAsequences were 120 s, 60 s and 60 s, respectively. It should however benoted that the capping step in the synthesis of the phosphorothioateDNA, sequence was performed after the oxidative sulfuration step, whichwas effected using 0.05 M 3H-1,2-benzodithiol-3-one 1,1-dioxide in MeCN;the standard 0.02 M iodine solution in THF/pyridine/water is employed inthe oxidation step of native DNA sequences. The last coupling reactionof each synthesis was performed using any of the activateddeoxyribonucleoside phosphoramidites 9a-d over a period of 180 s. TheLCAA-CPG-linked DNA sequence was then transferred to a 4-mL glass vialto which was added concentrated aqueous ammonia (1 mL). Thetightly-capped glass vial was placed in a heat block and kept at 65° C.for 16 h. The ammoniacal solution was transferred to another 4-mL glassvial and evaporated to half its original volume using a stream of air.

Example 7

This example demonstrates a solid-phase capture of DNA sequences inaccordance with an embodiment of the invention.

The aminooximated solid support 3 (150 mg) was washed with 20%triethylamine in MeCN (1 mL), filtered, blow dried under argon andplaced into a 1-mL glass vial. The above solution of unpurified5′-modified phosphorothioate or native DNA sequence 10a or 10e (˜500 μL)was added to the glass vial along with tetra-n-butylammonium chloride(14mg, 50 μmol); the solid-phase capture of the DNA sequence 10a or 10e wascarried out over a period of 3 h at 65° C. Near complete capture wasachieved upon oximation of the DNA sequence mediated by support 3 toproduce the silica gel support 11a or 11e. RP-HPLC analysis of thecapture reaction mixture confirmed the near absence of the DNA sequence10a or 10e (See FIG. 1B or FIG. 5B). Solid-phase capture of the DNAsequences 10b-d and 10f was performed as described for 10a. RP-HPLCanalysis of the capture reaction mixtures also confirmed the nearabsence of the DNA sequences 10b-d and 10f (see FIGS. 2B, 3B, 4B, and6B) thereby indicating near complete oximation of those sequences by thesupport 3. FIG. 4A shows the RP-HPLC profile of unpurified 10d(5′-functionalized 12d) spiked with a 14-, 16- and a 18-merphosphorothioate DNA sequence. FIG. 4B shows the RP-HPLC profile of thephosphorothioate DNA sequence 10d after capture by the support 3. FIG.4C shows the RP-HPLC analysis of solid-phase purified 12d that has beenreleased from the support 11d. FIG. 4D (middle lane) shows the purityanalysis of the solid-phase purified 12d by PAGE. FIGS. 1A, 2A, 3A, 5A,and 6A show the RP-HPLC profiles of unpurified 10a, 10b, 10c, 10e, and10f (5′-functionalized 12a, 12b, 12c, 12e, and 12f), respectively. FIGS.1B, 2B, 3B, 5B, and 6B show the RP-HPLC profiles of remaining unpurified10a, 10b, 10c, 10e, and 10f, respectively, after capture by support 3.FIGS. 1C, 2C, 3C, 5C, and 6C show the RP-HPLC analysis of solid-phasepurified 12a, 12b, 12c, 12e, and 12f, that have been released from thesupports 11a, 11b, 11c, lle, and llf respectively. FIGS. 1D, 2D, 3D, 5D,and 6D show the purity analysis of the solid-phase purified 12a, 12b,12c, 12e, and 12f, respectively, by PAGE.

Example 8

This example demonstrates a release of the DNA sequences from thesupports 11a-f in accordance with an embodiment of the invention.

The support 11a-f was placed in a 4 mL-screw-capped glass vial to whichis added a solution of concd. Aq. NH₃:MeCN (1:1 v/v) (1 mL); the glassvial was heated at 65° C. for 30 min and then subjected to filtration.This wash step was repeated once more under identical conditions and isfollowed by five DMSO washes (1-mL each). Release of the purified DNAsequence from 11a-f was effected by treatment with 1.0 M TBAF in dryDMSO (0.5 mL) in a sealed glass vial kept at 65° C. over a period of 3h. Methoxytrimethylsilane (200 μL) and MeCN (200 μL) were added to thesuspension, which was kept at 25° C. for 30 min and was then filteredthrough a sintered glass funnel; the filtrate was collected. The solidsupport was suspended in a solution of concentrated aqueous NH₃:MeCN(1:1 v/v) (1 mL) and heated at 65° C. for 30 min and filtered againthrough a glass-sintered funnel; the filtrate was collected and combinedwith the previous filtrate. The process was repeated once more underidentical conditions. All collected filtrates were pooled together andconcentrated under vacuum to approximately 100 μL. THF (1 mL) was addedto the concentrated solution in order to precipitate the DNA sequence12a-f. The precipitate was centrifuged at 14,000×g for 15 min at 25° C.;the supernatant is then carefully removed by suction. The DNA pellet waswashed with THF (3×1 mL). The pure DNA sequence 12a-f was dried underreduced pressure and stored at −20° C. until further use. The DNAsequence 12a-f has been characterized by mass spectrometry and itspurity by both RP-HPLC and PAGE.

12a: (SEQ ID NO. 7)5′-d(A_(PS)C_(PS)A_(PS)C_(PS)T_(PS)G_(PS)T_(PS)G_(PS)A_(PS)A_(PS)T_(PS)C_(PS)G_(PS)A_(PS)T_(PS)G_(PS)C_(PS)C_(PS)A_(PS)T). -ESI-MS: Calcd. 6406, found, 6407. 12b: (SEQ ID NO. 8)5′-d(C_(PS)T_(PS)C_(PS)C_(PS)G_(PS)T_(PS)A_(PS)C_(PS)C_(PS)T_(PS)T_(PS)A_(PS)C_(PS)G_(PS)T_(PS)C_(PS)T_(PS)T_(PS)G_(P)_(S)T). -ESI-MS: Calcd. 6315, found, 6315. 12c: (SEQ ID NO. 9)5′-d(G_(PS)T_(PS)G_(PS)A_(PS)G_(PS)T_(PS)A_(PS)G_(PS)C_(PS)G_(PS)A_(PS)A_(PS)C_(PS)G_(PS)T_(PS)G_(PS)A_(PS)A_(PS)G_(PS)T). -ESI-MS: Calcd. 6551, found, 6551. 12d: (SEQ ID NO. 10)5′d(T_(PS)A_(PS)T_(PS)C_(PS)C_(PS)G_(PS)T_(PS)A_(PS)G_(PS)C_(PS)T_(PS)A_(PS)A_(PS)C_(PS)G_(PS)T_(PS)C_(PS)A_(PS)G_(PS)T). -ESI-MS: Calcd. 6397, found, 6397. 12e: (SEQ ID NO. 12)5′-d(A_(P)C_(P)A_(P)C_(P)T_(P)G_(P)T_(P)G_(P)A_(P)A_(P)T_(P)C_(P)G_(P)A_(P)T_(P)G_(P)C_(P)C_(P)A_(P)T). -ESI-MS: Calcd. 6101, found, 6101.

Example 9

This example demonstrates a PAGE analysis of the solid-phase purifiedDNA sequences 12a-f in accordance with an embodiment of the invention.

An aqueous solution (0.25 OD₂₆₀) of each solid-phase purifiedphosphorothioate and native DNA sequences in a 1.5-mL microcentrifugetube, was evaporated to dryness under reduced pressure. To each tube wasadded 10 μL of loading buffer [10× Tris borate EDTA buffer (TBE), pH8.3, in formamide 1:4 (v/v) containing 2 mg/mL bromphenol blue]. Thesolution was then vigorously vortexed, centrifuged and loaded into a2-cm-wide well of a 20×40 cm, 7 M urea/20% polyacrylamide gel (12a-e) or7 M urea/18% polyacrylamide gel (12f). Electrophoresis was performed at375 V using 1× TBE buffer pH 8.3 as an electrolyte until the bromphenolblue dye traveled ˜80% the length of the gel. The electrophoresisapparatus was dismantled and the gel immersed in 250 mL of a solutioncomposed of i-PrOH:H₂O:formamide (2:8:0.4 v/v/v) to which was added a 1mg/mL Stains-All solution (10 mL) in formamide. The gel was agitated for3-4 h in the dark whereupon the staining solution was discarded and thegel washed with distilled water (3×250 mL). The gel was then exposed tonatural light until disappearance of the purple background and scanned.The DNA sequences appeared as blue (12e-f) or purple bands (12a-d)against a white background.

Example 10

This example demonstrates an enzymatic hydrolysis of the native DNAsequences 12e-f in accordance with an embodiment of the invention.

One OD260 unit of an aqueous solution of the solid-phase purified anddesalted native DNA sequence 12e or 12f was pipetted into amicrocentrifuge tube. The DNA solution was evaporated to dryness underreduced pressure whereupon 1.0 M Tris.Cl buffer pH 9.0 (6 μL), 1.0 MMgCl₂ (8 μL) and water (75 μL) are added followed by, after mixing,snake venom phosphodiesterase (Crotallus adamanteus, 0.015 U, 5 μL) andbacterial alkaline phosphatase (E. Coli, 0.7 U, 6 μL). The enzymaticreactions were allowed to proceed at 37° C. for 16 h. Deactivation ofthe enzymes was carried out by heating the digest at 90° C. for 3 min.The digest was centrifuged at 14,000×g for 5 min at 25° C. Immediatelyafter centrifugation, an aliquot (50 μL) of the digest was analyzed byRP-HPLC using a 5 μm Supelcosil LC-18S column (25 cm×4.6 mm) accordingto the following conditions: starting from 0.1 M triethylammoniumacetate pH 7.0, a linear gradient of 2.5% MeCN/min was pumped at a flowrate of 1 mL/min for 40 min. RP-HPLC chromatograms of the digests of 12eand 12f are shown in FIGS. 7A and 7B, respectively.

Example 11

This example demonstrates a determination of the efficiency of thesolid-phase purification process in accordance with an embodiment of theinvention.

To a RP-HPLC-purified and desalted DNA sequence (10a, 70 OD₂₆₀) in a1-mL glass vial was added a 0.1 M solution of tetra-n-butylammoniumchloride in H₂O (150 μL) and support 3 (70 mg). The suspension wasprocessed as described above (Examples 7 and 8) for the solid-phasepurification of unpurified 10a to give 12a after release from thesolid-support 11a and precipitation in THF. The efficiency of thesolid-phase purification process was determined from the followingequation: (OD₂₆₀ of the DNA sequence 12a recovered after the solid-phasepurification process ÷OD₂₆₀ of the DNA sequence 10a before solid-phasepurification)×100, that is (63 OD₂₆₀÷70 OD₂₆₀)100=90%.

Example 12

This example demonstrates a ten-fold scale up of the solid-phasepurification of the phosphorothioate DNA sequence 12a in accordance withan embodiment of the invention.

Ten individual solid-phase syntheses of the phosphorothioate DNAsequence 10a were each conducted on a scale of 1 μmole under conditionsidentical as those described above for the DNA sequences 10a-d (Example6). Upon completion of the syntheses, each individual DNA sequence wassubjected to a standard deprotection and release from the CPG supportprotocol under basic conditions. Upon complete deprotection and releaseof each DNA sequence from the CPG support, the individual ammoniacalsolutions of the ten DNA sequences were pooled together androtoevaporated to half its original volume (˜5 mL) under reducedpressure. A 0.1 M solution of tetra-n-butylammonium chloride in DMSO:H₂O(1:1 v/v) (5.0 mL) was added to the crude DNA solution. This solutionwas then added to a 20-mL glass vial containing the aminooximated solidsupport 3 (1.50 g), which had previously been washed with 20%trimethylamine in MeCN (10 mL), filtered and blow-dried under argon; theglass vial was tightly sealed and mechanically agitated over a period of3 h at 65° C. An aliquot (0.5 μL) of the capture reaction mixture wasanalyzed by RP-HPLC, which has only revealed the presence of benzamideand shorter than full-length DNA sequences relative to a similaranalysis performed prior to the capture of the crude DNA sequence 10a(FIGS. 10A-10C).

The solid support 11a was suspended in a solution of 10% Et₃N inMeCN:H₂O (1:1 v/v) (10 mL) and heated at 65° C. for 30 min whereupon thesuspension is filtered through a glass-sintered funnel. This process wasrepeated twice under identical conditions and was followed by multipleanhydrous DMSO (5×10 mL) washes. Release of the purified DNA sequencefrom 11a was effected by treatment with 1.0 M TBAF in dry DMSO (5 mL) ina sealed 20-mL glass vial kept at 65° C. over a period of 3 h.Methoxytrimethylsilane (2 mL) and MeCN (2 mL) were added to the reactionmixture, which was allowed to stand at 25° C. for 30 min in order toconsume unreacted fluoride ions. The suspension was filtered; the solidsupport was suspended in a solution of 10% Et₃N in MeCN:H₂O (1:1 v/v)(10 mL), heated at 65° C. for 30 min and then filtered through aglass-sintered funnel. This process was repeated once more underidentical conditions. All the post-release filtrates were pooledtogether and rotoevaporated under vacuum until approximately 1 mL of theoriginal volume is left. THF (20 mL) was then added to the filtrate; theDNA precipitate was centrifuged and the supernatant is carefully removedby suction. The DNA pellet was washed with THF (3×10 mL) and subjectedto identical centrifugation and supernatant removal conditions. The purenucleic acid sequence 12a was dried under reduced pressure and stored at−20° C. FIG. 10D shows the purity analysis of the solid-phase purified12a by PAGE.

Example 13

This example demonstrates a synthesis of an RNA linker compound and itsphosphoramidite derivative in accordance with an embodiment of theinvention.

To compound 6b of Scheme 2 (780 mg, 2.28 mmol) and imidazole (184 mg,2.70 mmol) in a flame-dried 25 mL-flask, was added under argon, dry DMF(5 mL) and N,N-diisopropylethylamine (2.35 mL,13.5 mmol); the solutionis then cooled to 0° C. Dichlorodiisopropylsilane (730 μL, 4.50 mmol)was added to the cold solution, which was left stirring for 1 h at 0° C.The reaction mixture was allowed to warm up to room temperature over aperiod of 4 h and then cooled to −60° C. A solution of2′-O-tert-butyldimethylsilyl uridine (1.93 g, 5.40 mmol) and imidazole(368 mg, 5.40 mmol) in dry DMF (5 mL) was added dropwise to the reactionmixture, which was kept stirring at −60° C. for 1 h. The reaction wasthen allowed to warm up to 0° C. and was left stirring for 3 h at thesame temperature. The reaction mixture was quenched by the addition ofcold (0° C.) 5% aq. NaHCO₃ (40 mL) and EtOAc (40 mL); after vigorousshaking, the organic layer was collected and rotoevaporated to drynessunder low pressure. The crude product was dissolved in a minimal volumeof CHCl₃ (4 mL) and loaded on the top of a glass column packed withsilica gel (˜40 g) pre-equilibrated in CHCl₃. The product 13, thestructure of which is shown below, eluted from the column using agradient of MeOH (0→6%) in CHCl₃. Pure product (1.14 g, 1.37 mmol) wasisolated as a solid in a yield of 60%. +ESI-HRMS: Calcd forC₃₉H₇₂N₄O₁₀Si₂Na [M+Na]+835.4679, Found 835.4699.

The RP-HPLC of compound 13 is shown in FIG. 9. The RP-HPLC analysis isperformed using a 5 μm Supelcosil LC-18S column (25 cm×4.6 mm) accordingto the following conditions: starting from 0.1 M triethylammoniumacetate (pH 7.0), a linear gradient of 5.0% MeCN/min is pumped at a flowrate of 1 mL/min for 40 min.

The 5′-silylated-2′-O-tert-butyldimethylsilyl uridine 13 (835 mg, 1.00mmol) was placed in a flame-dried 100 mL round-bottom flask anddissolved in anhydrous CH₂Cl₂ (10 mL). N,N-Diisopropylethylamine (536μ1, 3.00 mmol) was added to the solution followed by 2-cyanoethylN,N-diisopropylchlorophosphoramidite (335 μL,1.50 mmol). The reactionmixture was stirred at 25° C. for 5 h, quenched by the addition of H₂O(10 mL) and diluted with CH₂Cl₂ (25 mL). After vigorous shaking, theorganic layer was collected, dried over anhydrous Na₂SO₄ androtoevaporated under reduced pressure to afford an oil. The oilymaterial was evenly spread on the top of a column packed with silica gel(˜25 g) pre-equilibrated in benzene:Et₃N (9:1 v/v). The product, thestructure of which is shown below, eluted from the column usingbenzene:Et₃N (9:1 v/v) as the eluent. Fractions containing the productwere pooled together, concentrated under reduced pressure and dissolvedin dry benzene (7 mL). The solution was frozen and then lyophilizedunder high vacuum to afford triethylamine-free product 14 (828mg, 0.80mmol) as a colorless oil in 80% yield. +ESI-HRMS: Calcd forC₄₈H₈₉N₆O₁₁Psi₂Na [M+Na]+1035.5758, Found 1035.5793.

Example 14

Iminooxymethyl propanoate as a protecting group for the 2′-hydroxyposition on RNA.

A formidable challenge in the chemical synthesis of oligoribonucleotidesis to design a suitable 2′-hydroxy protecting group for ribonucleosides.A protecting group should optimally: (i) be easy to introduce; (ii)remain completely stable throughout the full assembly of the RNAsequence and particularly under the conditions used for nucleobase andphosphate deprotection and for the release of the sequence from thesolid support; (iii) be totally removable under conditions that do notcompromise the structural integrity of the RNA sequence. The 2′-hydroxyprotecting group should preferably be structurally flexible andsterically small enough to permit rapid phosphoramidite-couplingkinetics and high coupling efficiencies. Disclosed herein is animinooxymethyl group for 2′-hydroxy protection of ribonucleosides thatdemonstrates unprecedented cleavage through an innovative, entropydriven, intramolecular decarboxylative process.

-   Results and Discussion: As shown in Scheme 5, the usefulness of    2′-O-aminooxymethylribonucleosides 15a-d in the development of novel    2′-hydroxy protecting groups is further demonstrated by mixing    ribonucleoside 15a with ethyl pyruvate in the presence of drops of    concentrated HCl in MeOH.

-   After 1 hour at 25° C., 2′-O-iminooxymethyl ethyl propanate    derivative 16a was isolated after purification with silica gel in a    yield of 78%. Saponification of 16a upon treatment with NaOH (0.1 M)    produced 2′-O-iminooxymethyl propanoate salt 17a in quantitative    yield. Heating 17a in the presence of tetra-n-butylammonium fluoride    (TBAF) or chloride (TBACl) in dry dimethyl sulfoxide (DMSO)    (Scheme 5) resulted in a clean decarboxylation of 17a with the    concomitant formation of MeCN and formaldehyde to provide uridine    (19a) in quantitative yield based on RP-HPLC (reversed phase HPLC)    analysis of the reaction (FIG. 11C). FIGS. 11A-11D provide the    RP-HPLC profile for the silica-gel-purified 2′-O-protected uridine    16a (FIG. 11A); the de-esterified 2′-O-protected uridine 17a (FIG.    11B); and the 2′-O-deprotected uridine 19a (FIG. 11C), that is    compared to a commercial sample of uridine (FIG. 11D, control).    Uridine was used as an exemplary nucleoside, but the method is    generally applicable to all four ribonucleosides and the protecting    group is quantitatively removed when needed (FIGS. 12A-14D).

The solid-phase synthesis of a chimeric polyuridylic acid (U_(p))₂₀dT(FIGS. 15A-15C) served as a preliminary model for an assessment ofiminooxymethyl propanoate protection/deprotection method for use insolid-phase synthesis of RNA synthesis. First, the 5′-O-protection ofribonucleoside 16a upon reaction with 4,4′-dimethoxytrityl chloride indry pyridine produced 20 (Scheme 6) in a yield of 90% after purificationwith silica gel. The reaction of 20 with2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence oftriethylamine gives the ribonucleoside phosphoramidite 21 in apost-purification yield of 88%.

-   Synthesis of ribonucleoside phosphoramidite 21.-   The coupling kinetics and efficiency of ribonucleoside    phosphoramidite 21 were evaluated through the solid-phase synthesis    of (U_(p))₂₀dT (FIG. 15). An identical RNA control sequence was also    prepared by using a commercial uridine    2′-O-(tert-butyldimethylsilyl) phosphoramidite with the purpose of    comparing the quality of the RNA sequences produced from each    phosphoramidite monomer. Upon completion of the solid-phase RNA    syntheses, the RNA sequence constructed from 21 was subjected to    treatment with tetra-n-butylammonium hydroxide (0.5 M) for 3 h at    25° C. to concurrently de-esterify the 2′-O-protecting groups,    remove the 2-cyanoethyl-phosphate protecting groups, and release the    RNA sequence from the solid support. The basic nucleic acid solution    was then neutralized by adding a four molar equivalent of glacial    acetic acid and concentrated to dryness. Without isolation, the    crude RNA sequence was dissolved in a solution of TBAF or TBACl    (0.5 M) in dry DMSO and heated at 55° C. for 3 h to induce    decarboxylation of the 2′-O-iminooxypropanoate groups. The control    RNA sequence was fully deprotected and released from the support    under published conditions. Analytical samples of the unpurified and    desalted RNA sequences were analyzed by RP-HPLC and polyacrylamide    gel electrophoresis (PAGE) under denaturing conditions (FIGS.    15A-15C and 16, respectively). Moreover, enzymatic hydrolysis of the    crude, de-esterified, 2′-O-protected (U_(p))₂₀dT (FIG. 15A)    catalyzed by snake venom phosphodiesterase (SVP) and alkaline    phosphatase cleanly led to the production of 17a (Scheme 5) and    thymidine (FIG. 17A), whereas the enzymatic hydrolysis of crude,    de-esterified, 2′-O-protected (U_(p))₂₀dT revealed, after    decarboxylation, only the presence of uridine (19a, Scheme 5) and    thymidine (FIG. 17B), thereby indicating complete cleavage of the    2′-O-protecting groups from the chimeric RNA sequence.-   Materials and Methods: Common chemicals and solvents in addition to    DMSO, glacial acetic acid, acetic anhydride, hydrochloric acid,    aqueous ammonia, potassium carbonate, pyridine, triethylamine,    methanol, sodium hydroxide, concentrated hydrochloric acid,    Et₃N.3HF, DEPC-treated H₂O, sulfuryl chloride, N-hydroxyphthalimide,    1,8-diazabicyclo[5.4.01]-undec-7-ene (DBU), ammonium fluoride,    tetra-n-butylammonium fluoride(TBAF), tetra-n-butylammonium    chloride(TBACl), tetra-n-butylammonium hydroxide(TBAOH),    tetra-n-butylammonium acetate(TBAOAc), tetra-n-butylammonium    cyanide(TBACN), ethyl pyruvate, 4,4′-dimethoxytrityl chloride,    2-cyanoethyl N,N-diisopropylchlorophosphoramidite, and anhydrous    solvents (MeCN, CH₂Cl₂, C₆H₆, C₆D₆, pyridine, THF) and DMSO-d6 were    purchased from commercial sources and used without further    purification.    N4-phenoxyacetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine,    N6-phenoxyacetyl-3′,5′-O-(1,1,3,3-tetra-isopropyldisiloxane-1,3-diyl)adenosine,    N2-phenoxyacetyl-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)guanosine,    3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine,    5′-O-(4,4′-dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)uridine    phosphoramidite, PD-10 (Sephadex G-25M) columns,0.25 M    5-benzylthio-1H-tetrazole in MeCN or 0.25 M 5-ethylthio-1H-tetrazole    in MeCN and all ancillary reagents commonly used in solid-phase    DNA/RNA synthesis including succinyl long chain alkylamine    controlled-pore glass (CPG) support functionalized with    2′-deoxythymidine,as the leader nucleoside, were obtained from    reputable sources and used as received.

Reagent for enzymatic hydrolysis of RNA sequences such as magnesiumchloride, Tris.Cl, snake venom phosphodiesterase (Crotallus adamanteus)and bacterial alkaline phosphatase (E. coli) were bought from commercialsources and used without further purification. Flash chromatographypurifications were performed on glass columns (2.5 cm or 6.0 cm I.D.)packed with silica gel 60 (230-400 mesh), whereas analytical thin-layerchromatography (TLC) analyses were conducted on 2.5 cm×7.5 cm glassplates coated with a 0.25 mm thick layer of silica gel 60 F₂₅₄.Analytical RP-HPLC analyses were done using a 5 μm Supelcosil LC-18Scolumn (25 cm×4.6 mm) according to the following conditions: startingfrom 0.1 M triethylammonium acetate pH 7.0, a linear gradient of 1%MeCN/min was pumped at a flow rate of 1 mL/min for 40 min. In allRP-HPLC chromatograms, peak heights were normalized to the highest peak,which was set to 1 arbitrary unit. 2 M Triethylammonium acetate bufferwas purchased from Applied Biosystem and diluted to 0.1 M with HPLCgrade water prior to use.

Electrophoresis was conducted on a 20 cm×40 cm×0.75 mm 7 M-urea 20%polyacrylamide gel. The gel was run at 350 V, using lx Tris Borate EDTAas the electrolyte, until the bromphenol blue dye traveled 75%-80% ofthe gel's length. The gel was then immersed in 250 mL of Staining buffer(1:5:20 (v/v/v) formamide:isopropyl alcohol:ddH₂O) to which was added 10mL of Stains-all (1 mg/mL) in formamide. The gel was stained over about4 h in the dark. The staining solution was discarded and the gel wasrinsed three times with 250 mL distilled water. The gel was exposed tonatural light until the purple background disappeared. Unpurifiedde-esterified 2′-O-protected (Up)₂₀dT and fully 2′-O-deprotected(Up)₂₀dT appeared as a sharp purple or blue band, respectively. All NMRexperiments were performed using a spectrometer operating at 300.13,75.47 and 121.5 MHz for one-dimensional 1H,1H-decoupled ¹³C and¹H-decoupled ³¹P, respectively. Samples were maintained at a temperatureof 298 K. All spectra were recorded in deuterated solvents and chemicalshifts δ reported in parts per million (ppm) relative to appropriateinternal references.

High resolution and low resolution mass spectrometry analyses of newcompounds and RNA sequences were performed under contract at a reputablemass spectrometry facility.

General Procedure for the Synthesis of Ribonucleoside2′-O-Iminooxymethyl Propanoicacid Ethyl Esters (16a-d).

To a solution of3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2′-O-phthalimidooxymethyl)uridine(3.3 g, 5.0 mmol) in MeOH (25 mL) was added ammonium fluoride (1.8 g, 50mmol). The heterogeneous reaction mixture was stirred at about 25° C.until desilylation and dephthalimidation were complete (16 h) asindicated by TLC [CHCl₃:MeOH (9:1 v/v)]. To this mixture was added asolution (20 mL) of ethyl pyruvate (6.9 g, 60 mmol) in MeOH:H₂O (1:1,v/v) containing concentrated HCl (0.5 mL); the reaction mixture wasstirred over 1 h at 55° C. Approximately one half of the volatiles wereremoved by rotoevaporation under vacuum; the material was left was mixedwith dry silica gel (30 g) and allowed to dry overnight. The gel mix wasre-suspended in CH₂Cl₂ (30 mL) and layered on the top of achromatography column packed with silica gel (150 g) pre-equilibrated inCH₂Cl₂. The product was eluted from the column using a gradient of MeOH(0→6%) in CH₂Cl₂. Fractions containing the pure product were collectedand rotoevaporated under reduced pressure to give 16a (1.5 g, 3.9 mmol)as a white solid in a yield of 78%. The ribonucleoside2′-O-iminooxyethyl propanoic acid ethyl ester 16b-d were prepared in asimilar manner and were isolated with yields in the range of 61%-76%.16a: ¹H NMR (300 MHz, DMSO-d6) δ11.30 (br s, 1H), 7.85 (d, J=8.1 Hz,1H), 5.88 (d, J=5.6 Hz, 1H), 5.59 (dd, J=8.1, 2.2 Hz, 1H), 5.30 (m, 2H),5.22 (d, J=5.4 Hz, 1H), 5.13 (t, J=5.1 Hz, 1H), 4.30 (t, J=5.4 Hz, 1H),4.19 (q, J=7.1 Hz, 2H), 4.15 (m, 1H), 3.87 (m, 1H), 3.66-3.51 (m, 2H),1.95 (s, 3H), 1.24 (t, J=7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d6): δ162.9,162.8, 150.7, 150.5, 140.5, 101.8, 96.7, 85.9, 85.2, 79.2, 68.6, 61.4,60.7, 13.9, 11.6.+ESI-HRMS: Calcd. for C₁₅H₂₂N₃O₉ [M+H]⁺388.1351, Found:388.1349. 16b: ¹H NMR (300 MHz, DMSO-d6) δ9.29 (br s, 1H), 8.43 (br s,1H), 8.21 (d, J=7.8 Hz, 1H), 6.06 (d, J=7.8 Hz, 1H), 5.80 (d, J=3.7 Hz,1H), 5.37 (m, 2H), 5.28 (m, 1H), 4.30 (t, J=4.2 Hz, 1H), 4.22 (q, J=7.1Hz, 2H), 4.12 (t,J=5.2 Hz, 1H), 3.90 (m, 1H), 3.73-3.56 (m, 2H), 1.98(s, 3H), 1.25 (t, J=7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d6): δ162.9,160.7, 150.7, 148.7, 143.5, 96.9, 93.9, 87.7, 84.8, 79.8, 67.8, 61.4,59.8, 13.9, 11.7.+ESI-HRMS: Calcd. for C₁₅H₂₃N₄O₈ [M+H]⁺387.1510, Found:387.1511. 16c: ¹H NMR (300 MHz, DMSO-d6) δ8.29 (s, 1H), 8.08 (s, 1H),7.32 (br s, 2H), 5.99 (d, J=6.4 Hz, 1H), 5.27 (m, 2H), 4.88 (dd, J=6.3,6.1 Hz, 1H), 4.37 (m, 1H), 4.11 (dq, J=7.1, 1.5 Hz, 2H), 4.00 (q, J=3.1Hz, 1H), 3.62 (m, 2H), 1.64 (s, 3H), 1.19 (t, J=7.1 Hz, 3H). ¹³C NMR (75MHz, DMSO-d6): δ162.4, 156.1, 152.2, 150.3, 148.8, 139.9, 119.3, 96.6,86.4, 86.2, 79.1, 79.0, 69.2, 61.6, 61.2, 13.9, 11.0.+ESI-HRMS: Calcd.for C₁₆H₂₃N₆O₇ [M+H]⁺411.1623, Found: 411.1623.

16d: ¹H NMR (300 MHz, DMSO-d6) δ10.61 (s, 1H), 7.88 (s, 1H), 6.44 (br s,2H), S55.80 (d, J=6.4 Hz, 1H), 5.27 (m, 3H), 5.08 (m, 1H), 4.66 (dd,J=6.3, 6.1 Hz, 1H), 4.28 (m, 1H), 4.13 (q, J=7.1 Hz, 2H), 3.57 (m, 2H),1.78 (s, 3H), 1.21 (t, J=7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d6): δ162.6,156.6, 153.6, 151.2, 150.5, 135.3, 116.5, 96.8, 85.8, 84.4, 80.0, 69.3,61.4, 61.3, 13.9, 11.3.+ESI-HRMS: Calcd. forC₁₆H₂₂N₆O₈Na[M+Na]⁺449.1391, Found: 449.1396.

General Procedure for the Preparation of Ribonucleoside2′-O-Iminooxymethyl Propanoates (17a-d).

The ribonucleoside 16a (1 mg, about 2.6 μmol) was dissolved in aqueous0.1 M NaOH (1 mL). The solution was stirred at 25° C. over a period of 1h whereupon 1 M AcOH (0.5 mL) was added to neutralize the excess NaOH.The reaction mixture was then rotoevaporated to dryness under reducedpressure to afford 17a in quantitative yields as demonstrated by RP-HPLCanalysis of the saponification reaction (FIGS. 11A-11D). Saponificationof the ribonucleosides 16b-d was performed under similar conditions toprovide 17b-d in similar yields.

-   17a:-ESI-HRMS: Calcd. for C₁₃H₁₆N₃O₉[M]⁻358.0892, Found: 358.0886.-   17b:-ESI-HRMS: Calcd. for C₁₃H₁₇N₄O₈[M]⁻357.1052, Found: 357.1042.-   17c:-ESI-HRMS: Calcd. for C₁₄H₁₇N₆O₇[M]⁻381.1164, Found: 381.1167.-   17d:+ESI-HRMS: Calcd. for C₁₄H₁₇N₆O₈Na₂ [M+2Na]⁺443.0898, Found:    443.0900

General Procedure for the Decarboxylation of Ribonucleoside2′-O-Iminooxymethyl Propanoate 17a-d.

De-esterified 2′-O-protected uridine 17a (0.1 mg) was placed in a 4-mLscrew-capped glass vial. A solution of 0.5 M tetra-n-butylammoniumfluoride or tetra-n-butylammonium chloride in dry DMSO (0.5 mL) wasadded to the vial, which was then sealed and placed in a heat block keptat 55° C.; the reaction mixture was kept at this temperature for 60 min.As shown in FIGS. 11A-11D, RP-HPLC analysis of the decarboxylationreaction displayed complete disappearance of 17a and appearance ofuridine (19a) as the sole product. The decarboxylation of de-esterified2′-O-protected ribonucleosides (17b-d) was carried out under conditionsidentical to those described for the 2′-O-deprotection of 17a. FIGS. 12,13 and 14 showed quantitative decarboxylation of 17b-d and production of19b-d, as determined by RP-HPLC analysis of each 2′-O-deprotectionreaction. All decarboxylation reactions led to the quantitativeproduction of acetonitrile, as convincingly demonstrated by ¹³C-NMRanalysis of the 2′-O-deprotection of 17a (not shown).

Uridine 5′-O-(4,4′-dimethoxytrityl)-2′-O-iminooxymethyl propanoic acidethyl ester (20).

Uridine 2′-O-iminooxymethyl propanoic acid ethyl ester (16a, 1.2 g, 3.0mmol) was co-evaporated with anhydrous pyridine (2×20 ml) and thendissolved in anhydrous pyridine (20 mL). 4,4′-Dimethoxytrityl chloride(1.2 g, 3.6 mmol) was added to the solution, which was then allowed tostir for 3 h at about 25° C. The reaction mixture was rotoevaporatedunder reduced pressure to a gummy material, which was dissolved inCH₂Cl₂ (150 mL). The solution was subjected to extraction with asaturated aqueous NaHCO₃ solution (70 mL). The organic layer wascollected, dried over Na₂SO₄ and filtered. The filtrate wasrotoevaporated under reduced pressure; the material left was purified bychromatography on silica gel using a gradient of MeOH (0→2%) in CH₂Cl₂as the eluent to afford 20 as a solid (1.86 g, 2.70 mmol) in a yield of90%.

¹H NMR (300 MHz, DMSO-d6) δ11.37 (s, 1H), 7.67(d, J=8.1 Hz, 1H),7.39 (m,1H), 7.32 (m, 3H), 7.25 (m, 5H), 6.89 (d, J=8.4 Hz, 4H),5.81(d, J=4.1Hz, 1H), 5.40(m, 1H), 5.32(dd,J=7.8, 6.4 Hz,3H), 4.34(dd, J=4.8, 4.4 Hz,1H),4.22 (m, 1H),4.16(q, J=7.1 Hz, 2H), 3.96(m, 1H), 3.74(s, 6H), 1.95(s, 3H), 1.21(t, J=7.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d6): δ 162.86,162.82, 158.1, 150.7, 150.2, 149.6, 144.6, 140.6, 136.1, 135.3, 135.1,129.7, 127.9, 127.7, 126.8, 123.9, 113.2, 101.6, 97.1, 87.4, 85.8, 82.6,79.3, 68.5, 62.9, 61.3, 55.0, 13.9, 11.6.+ESI-HRMS: Calcd forC₃₆H₃₉N₃O₁₁Cs [M+Cs]⁺822.1634, Found 822.1638.

Uridine5′-O-(4,4′-dimethoxytrityl)-3-O-[N,N-diisopropylamino)(2-cyanoethyloxy)]phosphinyl-2′-O-iminooxymethylpropanoic acid ethyl ester (21).

To a solution of 20 (1.72g, 2.50 mmol) in anhydrous CH₂Cl₂ (30 mL) wasadded, under an argon atmosphere, Et3N (1.39 mL, 10.0 mmol) and2-cyanoethyl N,N-diisopropyl-chlorophosphoramidite (1.31 mL, 5.00 mmol).The reaction mixture was stirred at about 25° C. until completedisappearance of 20 (2 h) as indicated by TLC [C₆H₆:Et₃N (9:1 v/v)]. Thereaction mixture was then diluted with CH₂Cl₂ (50 mL) and extracted withH2O (10 mL). The organic layer was collected, dried over anhydrousNa₂SO₄ and filtered. The filtrate was rotoevaporated to dryness undervacuum. The crude phosphoramidite was purified by chromatography onsilica gel using C₆H₆:Et₃N (9:1 v/v) as the eluent. Fractions containingthe pure product were pooled together and rotoevaporated to drynessunder low pressure. The material left was dissolved in dry C₆H₆(3 mL);the resulting solution was added to cold (−78° C.) stirred hexane (100mL). The pure phosphoramidite precipitated immediately as a white solid.Hexane was removed by decantation; the wet precipitate was dissolved indry C₆H₆ (5 mL) and lyophilized under high vacuum. Et₃N-free 21 wasisolated as a white powder (1.95 g, 2.20 mmol) in a yield of 88%.

³¹P NMR (121 MHz, C₆D₆): δ150.5, 149.5.+ESI-HRMS: Calcd for C₄₅H₅₆N₅O₁₂P(M+H)⁺890.3736, Found 890.3744.

Solid-Phase Synthesis of Chimeric Polyuridylic Acid Sequences.

The solid phase synthesis of 5′-r(UUUUUUUUUUUUUUUUUU-UU)dTRU_(p))₂₀dTπwas conducted on a scale of 0.2 μmole in the “trityl-off” mode using asuccinyl long chain alkylamine controlled-pore glass (CPG) supportfunctionalized with 2′-deoxythymidine as the leader nucleoside. Thesynthesis was carried out using a DNA/RNA synthesizer and theribonucleoside phosphoramidite monomer 21, which was dissolved in dryMeCN to a concentration of 0.1 M. 5-Benzylthio-1H-tetrazole (0.25 M inMeCN) and all other ancillary reagents necessary for solid-phase RNAsynthesis were obtained from commercial sources. The reaction time foreach phosphoramidite coupling step was set to 3 min. The stepwisecoupling efficiency was determined to be 99±5% by measuring,spectrophotometrically at 498 nm, the ratio of the molar amount oftrityl cation released after the second and last synthesis cycles. Thededimethoxytritylation, capping and oxidation steps of any synthesiscycle were each performed over a period of 60 s. The control chimericRNA sequence (U_(p))₂₀dT was synthesized using commercial2′-O-(tert-butyldimethylsilyl)uridine phosphoramidite monomer under thecontrol of the same automated synthetic protocol.

Deprotection and Characterization of the Chimeric RNA Sequences.

The solid-phase-linked 5′-dedimethoxytritylated RNA sequence was placedinto a 4-mL screw-capped glass vial and subjected to treatment with 0.5M tetra-n-butylammonium hydroxide 100 μL) for 3 h at 25° C. in order tode-esterify the 2′-O-protecting groups, remove the 2-cyanoethylphosphate protective groups and release the RNA sequence from the solidsupport. The solution was neutralized by addition of 1 M acetic acid(200 μL) and then evaporated to dryness using a stream of air. Withoutisolation or further purification, the de-esterified 2′-O-protected RNAsequence was dissolved in a 0.5 M TBAF or TBACl solution in dry DMSO(500 μL) and heated to 55° C. over a period of 3 h to achieve completedecarboxylation of the 2′-O-imminooxypropanoate groups. The solution wasconcentrated to a volume of about 100 μL, diluted with about 900 μL ofDEPC-treated water and desalted using a PD-10 (Sephadex G-25M) column.Unpurified and desalted (U_(p))₂₀dT was eluted from the column usingDEPC-treated H₂O as the eluent. Fractions (1 mL) were collected andthose containing the RNA sequence (A₂₆₀) were pooled together foranalysis by RP-HPLC (FIG. 15B), polyacrylamide gel electrophoresis (FIG.16, lane 2) and for characterization by ESI mass spectrometry. Thecontrol RNA sequence (U_(p))₂₀dT was released from the support and fullydeprotected according to published protocols. -ESI-MS analysis ofde-esterified of 2′-O-protected (U_(p))₂₀dT: Calcd. forC₂₇₀H₂₉₄N₆₂O₂₂₅P₂₀[M-20H]⁻8647, Found 8650.-ESI-MS analysis of fully2′-O-deprotected (U_(p))₂₀dT: Calcd. for C₁₉₀H₂₁₄N₄₂O₁₆₅P₂₀[M-4H]⁴⁻6342, Found 6342.

Enzymatic Hydrolysis of the Chimeric RNA Sequences.

One OD₂₆₀ unit of each of the unpurified and desalted chimeric RNAsequence and control RNA sequence (U_(p))₂₀dT was pipetted into separatemicrocentrifuge tubes. The RNA solution of each tube was evaporated todryness using a stream of air. To each tube was added 1.0 M Tris.Clbuffer pH 9.0 (6 μL), 1.0 M MgCl₂ (8 μL) and water (75 μL) followed by,after mixing, snake venom phosphodiesterase (Crotallus adamanteus, 0.015U, 5 μL) and bacterial alkaline phosphatase (E. coli, 0.7 U, 6 μL). Theenzymatic reactions were allowed to proceed at 37° C. for 16 h.Deactivation of the enzymes was carried out by heating the digests at90° C. for 3 min. Each digest was centrifuged at 14,000 rpm for 5 min at25° C. An aliquot (50 μL) of each digest was analyzed by RP-HPLC using a5 μm Supelcosil LC-18S column under the following conditions: startingfrom 0.1 M triethylammonium acetate (pH 7.0), a linear gradient of 1%MeCN/min was pumped at a flow rate of 1 mL/min for 40 min. RP-HPLCchromatograms of the digests are shown in FIGS. 17A-17C.

V. Exemplary Embodiments

The following numbered paragraphs illustrate exemplary embodiments ofthe disclosed technology.

Paragraph 1. A method of purifying an oligonucleotide or anoligonucleotide analog composed of “b” nucleotides from a mixturecomprising the oligonucleotide or oligonucleotide analog and at leastone oligonucleotide or oligonucleotide analog composed of “a”nucleotides, wherein b≠a, the method comprising:

(i) providing a protected nucleoside or nucleoside analog of formula (I)or (Ia) functionalized with an activatable phosphorus-containing entity

(ii) providing a mixture comprising an optionally protected firstoligonucleotide or oligonucleotide analog V′ composed of b-1 nucleotidesor nucleotide analogs and having a free 5′-terminal OH group, whereinthe first oligonucleotide V′ comprises phosphate or phosphorothioatetriester linkages, or a combination thereof, and wherein the firstoligonucleotide V′ is linked at its 3′-terminus to a solid support,wherein at least one oligonucleotide or oligonucleotide analog composedof “a” nucleotides is also linked to the solid support;

(iii) coupling the first oligonucleotide V′ with the protectednucleoside or nucleoside analog of formula (I) or (Ia), to provide asecond oligonucleotide of the formula (II) or (IIa)

(iv) oxidizing or sulfurizing, optionally deprotecting, and cleaving thesecond oligonucleotide or oligonucleotide analog of the formula (II) or(IIa) from the solid support to form a mixture comprising a thirdoligonucleotide of the formula (III) or (IIIa)

wherein V is the moiety resulting after optional deprotection of thesecond oligonucleotide or oligonucleotide analog and wherein V is notlinked to the solid support;

(v) reacting the mixture comprising the third oligonucleotide oroligonucleotide analog of the formula (III) or (IIIa) with asilica-attached linker compound of the formula:

wherein A, K, and L are independently C₂-C₁₀ alkanediyl and R⁶ is H orC₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl, and wherein

is silica, to form a linker-attached oligonucleotide or oligonucleotideanalog of the formula (IV) or (IVa)

(vi) washing the linker-attached oligonucleotide of the formula (IV) or(IVa) with at least one solvent or a mixture of solvents to remove theoligonucleotide(s) or oligonucleotide analog(s) composed of “a”nucleotides;

(vii) treating the linker-attached oligonucleotide or oligonucleotideanalog of formula (IV) or (IVa) with a desilylation agent; and

(viii) isolating the purified oligonucleotide or oligonucleotide analogcomposed of “b” nucleotides from the product of step (vii);

wherein

B is an optionally protected nucleobase or an optionally protectednucleobase analog;

D and E are independently C₂-C₁₀ alkanediyl;

n is 1 to 4;

R¹ is C₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl;

R² is hydrogen or linear or branched C₁-C₆ alkyl;

R³ is linear or branched C₃-C₆ alkyl;

J is H or OR⁷ wherein R⁷ is a reversible or permanent hydroxylprotecting group;

X is O or S;

Y is H or C₁-C₆ linear alkyl;

W is a lone pair of electrons or an oxo function;

when W is a lone pair of electrons, Z is NR⁴R⁵ wherein R⁴ and R⁵ areindividually C₁-C₆ alkyl, C₆-C₁₀ arylated C₁-C₆ alkyl, or C₃-C₈cycloalkyl or R⁴ and R⁵ taken together with the nitrogen atom to whichthey are attached form a saturated 3-10 membered heterocyclic ringoptionally including one or more additional heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur, and Q is OT whereinT is a reversible or permanent hydroxyl protecting group; and

when W is an oxo function, Z is H and Q is O⁻.

Paragraph 2. The method of paragraph 1, wherein A is 1,5-pentanediyl.

Paragraph 3. The method of paragraph 1 or 2, wherein K is1,3-propanediyl.

Paragraph 4. The method of any one of paragraphs 1-3, wherein L is1,3-propanediyl.

Paragraph 5. The method of any one of paragraphs 1-4, wherein D is1,2-ethanediyl.

Paragraph 6. The method of any one of paragraphs 1-5, wherein E is1,5-pentanediyl.

Paragraph 7. The method of any one of paragraphs 1-6, wherein n is 1.

Paragraph 8. The method of any one of paragraphs 1-7, wherein R² ismethyl.

Paragraph 9. The method of any one of paragraphs 1-8, wherein R³ is2-propyl.

Paragraph 10. The method of any one of paragraphs 1-9, wherein R⁶ ismethyl.

Paragraph 11. The method of any one of paragraphs 1-10, wherein J is H.

Paragraph 12. The method of any one of paragraphs 1-10, wherein J isOR⁷.

Paragraph 13. The method of paragraph 12, wherein J is an acetal, ketal,thioacetal, or acetalester.

Paragraph 14. The method of paragraph 12, wherein R⁷ has a formula

wherein R¹⁰ is H, alkyl, cycloalkyl, or OR¹⁰ is O⁻M⁺.

Paragraph 15. The method of paragraph 13, wherein R¹⁰ is C₁₋₆alkyl.

Paragraph 16. The method of paragraph 14, wherein R¹⁰ is ethyl.

Paragraph 17. The method of any one of paragraphs 1-16, wherein Y ismethyl.

Paragraph 18. The method of any one of paragraphs 1-17, wherein thedesilylation agent comprises fluoride ion.

Paragraph 19. The method of any one of paragraphs 1-18, wherein theprotected nucleoside or nucleoside analog is of formula (I).

Paragraph 20. The method of any one of paragraphs 1-19, wherein W is alone pair of electrons.

Paragraph 21. The method of any one of paragraphs 1-20, wherein Z isNR⁴R⁵ wherein R⁴ and R⁵ are individually C₁-C₆ alkyl, C₆-C₁₀ arylatedC₁-C₆ alkyl, and C₃-C₈ cycloalkyl containing 1 to 10 carbon atoms or R⁴and R⁵ taken together with the nitrogen atom to which they are attachedform a saturated 3-10 membered heterocyclic ring optionally includingone or more additional heteroatoms selected from the group consisting ofnitrogen, oxygen and sulfur.

Paragraph 22. The method of any one of paragraphs 1-21, wherein Q is OTwherein T is a reversible or permanent hydroxyl protecting group.

Paragraph 23. The method of any one of paragraphs 1-22, wherein theoptionally protected first oligonucleotide or oligonucleotide analog issynthesized by a solid-phase protocol.

Paragraph 24. A compound of the formula:

wherein B is an optionally protected nucleobase or nucleobase analog,

D and E are independently C₂-C₁₀ alkanediyl,

n is 1 to 4,

R¹ is C₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl,

R² is hydrogen or C₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl,

R³ is linear or branched C₃-C₆ alkyl,

J is H or OR⁷ wherein R⁷ is a reversible or permanent hydroxylprotecting group,

X is O or S,

Y is H or linear C₁-C₆ alkyl,

W is a lone pair of electrons, and

Z is NR⁴R⁵ wherein R⁴ and R⁵ are individually C₁-C₆ alkyl, C₆-C₁₀arylated C₁-C₆ alkyl, or C₃-C₈ cycloalkyl containing 1 to 10 carbonatoms or R⁴ and R⁵ taken together with the nitrogen atom to which theyare attached form a saturated 3-10 membered heterocyclic ring optionallyincluding one or more additional heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur, and Q is OT wherein T is areversible or permanent hydroxyl protecting group.

Paragraph 25. The compound of paragraph 24, wherein R¹ is methyl.

Paragraph 26. The compound of paragraph 24 or paragraph 25, wherein R²is methyl.

Paragraph 27. The compound of any one of paragraphs 24-26, wherein R³ is2-propyl.

Paragraph 28. The compound of any one of paragraphs 24-27, wherein D is1,2-ethanediyl.

Paragraph 29. The compound of any one of paragraphs 24-28, wherein E is1,5-pentanediyl.

Paragraph 30. The compound of any one of paragraphs 24-29, wherein n is1.

Paragraph 31. The compound of any one of paragraphs 24-30, wherein J isH.

Paragraph 32. The compound of any one of paragraphs 24-31 wherein J isOR⁷ and R⁷ is a reversible or permanent hydroxyl protecting group stableto the alkaline conditions utilized in nucleobase and optionalphosphate/thiophosphate deprotection.

Paragraph 33. The compound of any one of paragraphs 24-32, wherein J isan acetal, ketal, thioacetal, or acetalester.

Paragraph 34. The compound of any one of paragraphs 24-33, wherein J isOR⁷, and R⁷ has a formula

wherein R¹⁰ is H, alkyl, cycloalkyl, or OR¹⁰ is O⁺M⁺.

Paragraph 35. The method of paragraph 34, wherein R¹⁰ is C₁₋₆alkyl.

Paragraph 36. The method of paragraph 35, wherein R¹⁰ is ethyl.

Paragraph 37. The compound of any one of paragraphs 24-36, wherein Z isdiisopropylamino.

Paragraph 38. A method of preparing the compound of any one ofparagraphs 24-37 wherein W is a lone pair of electrons, Z is NR⁴R⁵, andQ is OT wherein T is 2-cyanoethyl, comprising:

(i) providing a compound of the formula (1):

(ii) reacting the compound of step (i) with a compound of the formula:

R¹HN-D-NHR¹

to provide a compound of the formula (2):

(iii) reacting the compound of formula (2) with a compound of theformula:

to provide a compound of formula (3):

(iv) reacting the compound of formula (3) with a compound of theformula: (R³)₂SiX₂, wherein X is a leaving group, to provide a compoundof formula (4):

(v) reacting the compound of formula (4) with a nucleobase protected2′-deoxyribonucleoside or nucleobase-protected 2′-O-protectedribonucleoside or a nucleobase-protected and carbohydrate modifiedanalog thereof to provide a compound of the formula (5):

and

(vi) reacting the compound of formula (5) with [(R⁴R⁵)]₂NPOCH₂CH₂CN orR⁴R⁵NP(X′)OCH₂CH₂CN wherein X′ is a monovalent leaving group to providea compound of the formula (6):

Paragraph 39. A capture support of the formula (9):

wherein A, K, and L are independently C₂-C₁₀ alkanediyl and R⁶ is H orC₁-C₆ linear or branched alkyl or C₁-C₆ cycloalkyl, and wherein

is silica.

Paragraph 40. The capture support of paragraph 39, wherein R⁶ is methyl.

Paragraph 41. The capture support of paragraph 39 or 40, wherein A is1,5-pentanediyl.

Paragraph 42. The capture support of any one of paragraphs 39-41,wherein K and L are individually 1,3-propanediyl.

Paragraph 43. A method of preparing the capture support of any one ofparagraphs 39-42, comprising:

(i) providing a functionalized silica gel of the formula (10):

(ii) reacting the functionalized silica gel of the formula (10) with acompound of the formula:

to provide a compound of formula (11):

and

(iii) reacting the compound of formula (11) with a compound of theformula:

H₂NO-L-ONH₂

to provide the capture support of any one of paragraphs 39-42.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A method of purifying an oligonucleotide or an oligonucleotide analogcomposed of “b” nucleotides from a mixture comprising theoligonucleotide or oligonucleotide analog and at least oneoligonucleotide or oligonucleotide analog composed of “a” nucleotides,wherein b a, the method comprising: (i) providing a protected nucleosideor nucleoside analog of formula (I) or (Ia) functionalized with anactivatable phosphorus-containing entity

(ii) providing a mixture comprising an optionally protected firstoligonucleotide or oligonucleotide analog V′ composed of b-1 nucleotidesor nucleotide analogs and having a free 5′-terminal OH group, whereinthe first oligonucleotide V′ comprises phosphate or phosphorothioatetriester linkages, or a combination thereof, and wherein the firstoligonucleotide V′ is linked at its 3′-terminus to a solid support,wherein at least one oligonucleotide or oligonucleotide analog composedof “a” nucleotides is also linked to the solid support; (iii) couplingthe first oligonucleotide V′ with the protected nucleoside or nucleosideanalog of formula (I) or (Ia), to provide a second oligonucleotide ofthe formula (II) or (IIa)

(iv) oxidizing or sulfurizing, optionally deprotecting, and cleaving thesecond oligonucleotide or oligonucleotide analog of the formula (II) or(IIa) from the solid support to form a mixture comprising a thirdoligonucleotide of the formula (III) or (IIIa)

wherein V is the moiety resulting after optional deprotection of thesecond oligonucleotide or oligonucleotide analog and wherein V is notlinked to the solid support; (v) reacting the mixture comprising thethird oligonucleotide or oligonucleotide analog of the formula (III) or(IIIa) with a silica-attached linker compound of the formula:

wherein A, K, and L are independently C₂-C₁₀ alkanediyl and R⁶ is H orC₁-C₆ linear or branched alkyl or C₃-C₈ cycloalkyl, and wherein

is silica, to form a linker-attached oligonucleotide or oligonucleotideanalog of the formula (IV) or (IVa)

(vi) washing the linker-attached oligonucleotide of the formula (IV) or(IVa) with at least one solvent or a mixture of solvents to remove theoligonucleotide(s) or oligonucleotide analog(s) composed of “a”nucleotides; (vii) treating the linker-attached oligonucleotide oroligonucleotide analog of formula (IV) or (IVa) with a desilylationagent; and (viii) isolating the purified oligonucleotide oroligonucleotide analog composed of “b” nucleotides from the product ofstep (vii); wherein B is an optionally protected nucleobase or anoptionally protected nucleobase analog; D and E are independently C₂-C₁₀alkanediyl; n is 1 to 4; R¹ is C₁-C₆ linear or branched alkyl or C₃-C₈cycloalkyl; R² is hydrogen or linear or branched C₁-C₆ alkyl; R³ islinear or branched C₃-C₆ alkyl; J is H or OR⁷ wherein R⁷ is a reversibleor permanent hydroxyl protecting group; X is O or S; Y is H or C₁-C₆linear alkyl; W is a lone pair of electrons or an oxo (═O) function;when W is a lone pair of electrons, Z is NR⁴R⁵ wherein R⁴ and R⁵ areindividually C₁-C₆ alkyl, C₆-C₁₀ arylated C₁-C₆ alkyl, or C₃-C₈cycloalkyl or R⁴ and R⁵ taken together with the nitrogen atom to whichthey are attached form a saturated 3-10 membered heterocyclic ringoptionally including one or more additional heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur, and Q is OT whereinT is a reversible or permanent hydroxyl protecting group; and when W isan oxo function, Z is H and Q is
 2. The method of claim 1, wherein: A is1,5-pentanediyl; K is 1,3-propanediyl; L is 1,3-propanediyl; D is1,2-ethanediyl; E is 1,5-pentanediyl; R² is methyl; R³ is 2-propyl; R⁶is methyl; Y is methyl; n is 1; or a combination thereof. 3-11.(canceled)
 12. The method of claim 1, wherein A is 1,5-pentanediyl, K is1,3-propanediyl, L is 1,3-propanediyl, D is 1,2-ethanediyl, E is1,5-pentanediyl, n is 1, R² is methyl, R³ is 2-propyl, R⁶ is methyl, andY is methyl.
 13. The method of claim 1, wherein J is H.
 14. The methodof claim 1, wherein J is OR⁷.
 15. The method of claim 14, wherein J isan acetal, ketal, thioacetal, or acetalester.
 16. The method of claim15, wherein R⁷ has a formula

wherein R¹⁰ is H, alkyl, cycloalkyl, or OR¹⁰ is O⁻M⁺.
 17. The method ofclaim 16, wherein R¹⁰ is C₁₋₆alkyl.
 18. The method of claim 16, whereinR¹⁰ is ethyl.
 19. The method of claim 1, wherein the desilylation agentcomprises fluoride ion.
 20. The method of claim 1, wherein the protectednucleoside or nucleoside analog is of formula (I).
 21. The method ofclaim 20, wherein W is a lone pair of electrons.
 22. The method of claim21, wherein Z is NR⁴R⁵ wherein R⁴ and R⁵ are individually C₁-C₆ alkyl,C₆-C₁₀ arylatedC₁-C₆ alkyl, and C₃-C₈ cycloalkyl containing 1 to 10carbon atoms or R⁴ and R⁵ taken together with the nitrogen atom to whichthey are attached form a saturated 3-10 membered heterocyclic ringoptionally including one or more additional heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur.
 23. The method ofclaim 22, wherein Q is OT wherein T is a reversible or permanenthydroxyl protecting group.
 24. The method of claim 1, wherein theoptionally protected first oligonucleotide or oligonucleotide analog issynthesized by a solid-phase protocol.
 25. A compound of the formula:

wherein B is an optionally protected nucleobase or nucleobase analog, Dand E are independently C₂-C₁₀ alkanediyl, n is 1 to 4, R¹ is C₁-C₆linear or branched alkyl or C₃-C₈ cycloalkyl, R² is hydrogen or C₁-C₆linear or branched alkyl or C₃-C₈ cycloalkyl, R³ is linear or branchedC₃-C₆ alkyl, J is H or OR⁷ wherein R⁷ is a reversible or permanenthydroxyl protecting group, X is O or S, Y is H or linear C₁-C₆ alkyl, Wis a lone pair of electrons, and Z is NR⁴R⁵ wherein R⁴ and R⁵ areindividually C₁-C₆ alkyl, C₆-C₁₀ arylated C₁-C₆ alkyl, or C₃-C₈cycloalkyl containing 1 to 10 carbon atoms or R⁴ and R⁵ taken togetherwith the nitrogen atom to which they are attached form a saturated 3-10membered heterocyclic ring optionally including one or more additionalheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, and Q is OT wherein T is a reversible or permanent hydroxylprotecting group.
 26. The compound of claim 25, wherein: R¹ is methyl;R² is methyl; R³ is 2-propyl; or a combination thereof. 27-28.(canceled)
 29. The compound of claim 25, wherein: D is 1,2-ethanediyl; Eis 1,5-pentanediyl; or a combination thereof.
 30. (canceled)
 31. Thecompound of claim 25, wherein n is
 1. 32. The compound of claim 25,wherein Z is diisopropylamino.
 33. The compound of claim 25, wherein R¹is methyl, R² is methyl, R³ is 2-propyl, D is 1,2-ethanediyl, E is1,5-pentanediyl, n is 1, and Z is diisopropylamino.
 34. The compound ofclaim 25, wherein J is H.
 35. The compound of claim 25 wherein J is OR⁷and R⁷ is a reversible or permanent hydroxyl protecting group stable tothe alkaline conditions utilized in nucleobase and optionalphosphate/thiophosphate deprotection.
 36. The compound of claim 35,wherein J is an acetal, ketal, thioacetal, or acetalester.
 37. Thecompound of claim 35, wherein J is OR⁷, and R⁷ has a formula

wherein R¹⁰ is H, alkyl, cycloalkyl, or OR¹⁰ is O⁺M⁺.
 38. The method ofclaim 37, wherein R¹⁰ is C₁₋₆alkyl.
 39. The method of claim 38, whereinR¹⁰ is ethyl.
 40. A method of preparing the compound of claim 1 whereinW is a lone pair of electrons, Z is NR⁴R⁵, and Q is OT wherein T is2-cyanoethyl, comprising: (i) providing a compound of the formula (1):

(ii) reacting the compound of step (i) with a compound of the formula:R¹HN-D-NHR¹ to provide a compound of the formula (2):

(iii) reacting the compound of formula (2) with a compound of theformula:

to provide a compound of formula (3):

(iv) reacting the compound of formula (3) with a compound of theformula: (R³)₂SiX₂, wherein X is a leaving group, to provide a compoundof formula (4):

(v) reacting the compound of formula (4) with a nucleobase protected2′-deoxyribonucleoside or nucleobase-protected 2′-O-protectedribonucleoside or a nucleobase-protected and carbohydrate modifiedanalog thereof to provide a compound of the formula (5):

and (vi) reacting the compound of formula (5) with [(R⁴R⁵)]₂NPOCH₂CH₂CNor R⁴R⁵NP(X′)OCH₂CH₂CN wherein X′ is a monovalent leaving group toprovide a compound of the formula (6):


41. A capture support of the formula (9):

wherein A, K, and L are independently C₂-C₁₀ alkanediyl and R⁶ is H orC₁-C₆ linear or branched alkyl or C₁-C₆ cycloalkyl, and wherein

is silica. 42-44. (canceled)
 45. A method of preparing the capturesupport of claim 41, comprising: providing a functionalized silica gelof the formula (10):

(ii) reacting the functionalized silica gel of the formula (10) with acompound of the formula:

to provide a compound of formula (11):

and (iii) reacting the compound of formula (11) with a compound of theformula:H₂NO-L-ONH₂ to provide the capture support of claim 41.